Antibodies to lipoproteins and apolipoproteins and methods of use thereof

ABSTRACT

Compositions and methods using antibodies which are immunoreactive with specific apolipoproteins to determine the concentrations of lipoproteins such as HDL and LDL, and/or apolipoproteins in human blood, serum or plasma sample, are described. Monoclonal antibodies (MAbs) are described that specifically bind to epitopes present in apolipoproteins and lipoproteins, enabling rapid and reliable determinations of levels of specific blood lipoprotein and/or apolipoprotein levels, including Apo B-100, Apo A-I, Apo A-II, Apo C-III, and Apo E, and thereby determination of relative ratios of HDL and LDL and LpaI and LpaII. In a preferred embodiment, the compositions are strips of a solid phase material coated with one or more of the antibodies and are referred to herein as “dipsticks”. The dipsticks specifically bind a lipoprotein or apolipoprotein when dipped into a protein sample. The amount of lipid associated with a bound lipoprotein or the amount of apolipoprotein bound on the dipstick is quantitated using an appropriate method, for example, by staining with a lipid stain or reaction with a second labelled antibody. The intensity of the stain on the dipstick is proportional to the concentration of the lipoprotein lipid or apolipoprotein circulating in the blood and can be quantitated by comparison with standards containing known amounts of lipid.

This application is a continuation of U.S. Ser. No. 08/268,809 now U.S.Pat. No. 6,107,045 filed Jun. 30, 1994.

BACKGROUND OF THE INVENTION

This invention is generally in the field of methods and compositions forthe determination and quantitation of lipoproteins and apolipoproteinsin human blood.

Human Plasma Lipoproteins and Apolipoproteins

Plasma lipoproteins are carriers of lipids from the sites of synthesisand absorption to the sites of storage and/or utilization. Lipoproteinsare spherical particles with triglycerides and cholesterol esters intheir core and a layer of phospholipids, nonesterified cholesterol andapolipoproteins on the surface. They are categorized into five majorclasses based on their hydrated density as very large, triglyceride-richparticles known as chylomicrons (less than 0.95 g/ml), very low densitylipoproteins (VLDL, 0.95 to 1.006 g/ml), intermediate-densitylipoproteins (IDL, 1.006 to 1.019 g/ml), low-density lipoproteins (LDL,1.019 to 1.063 g/ml) and, high-density lipoproteins (HDL, 1.063 to 1.210g/ml). Plasma lipoproteins can be also classified on the basis of theirelectrophoretic mobility. HDL co-migrate with α-globulins, LDL withβ-globulins, VLDL between α- and β-globulins with so called pre-βglobulins, whereas chylomicrons remain at the point of application.(Osborne, J. D. and Brewer, B. Jr. Adv. Prot. Chem. 31:253–337 (1977);Smith, L. C. et al. Ann. Rev. Biochem., 47:751–777 (1978)).

Apolipoproteins are protein components of lipoproteins with three majorfunctions: (1) maintaining the stability of lipoprotein particles, (2)acting as cofactors for enzymes that act on lipoproteins, and (3)removing lipoproteins from circulation by receptor-mediated mechanisms.The four groups of apolipoproteins are apolipoproteins A (Apo A), B (ApoB), C (Apo C) and E (Apo E). Each of the three groups A, B and Cconsists of two or more distinct proteins. These are for Apo A: Apo A-I,Apo A-II, and Apo A-IV, for Apo B: Apo B-100 and Apo B-48; and for ApoC: Apo C-I, Apo C-II and Apo C-III. Apo E includes several isoforms.

Each class of lipoproteins includes a variety of apolipoproteins indiffering proportions with the exception of LDL, which contains ApoB-100 as a sole apolipoprotein. Apo A-I and Apo A-II constituteapproximately 90 percent of the protein moiety of HDL whereas Apo C andApo E are present in various proportions in chylomicrons, VLDL, IDL andHDL. Apo B-100 is present in LDL, VLDL and IDL. Apo B-48 resides only inchylomicrons and so called chylomicron remnants (Kane, J. P., Method.Enzymol. 129:123–129 (1986)).

Lipoprotein metabolism is a very complex process involving exogenous andendogenous pathways as well as a reverse cholesterol transport. In theexogenous pathway, the triglycerides and cholesterol from anindividual's diet are incorporated into chylomicrons which enter intothe blood stream via intestinal lymph. Lipoprotein lipase hydrolyzes thetriglyceride component of chylomicrons into free fatty acids which aretaken up by muscle cells and/or adipocytes. As the triglyceride core ofchylomicrons is depleted, chylomicron remnant particles are formed andremoved from the circulation via chylomicron remnant receptor present onthe surface of hepatic cells.

In the endogenous pathway, the liver synthesizes triglycerides andcholesterol. The endogenously made triglycerides and cholesterol arepacked into triglyceride rich VLDL particles and secreted into thecirculation. Once in the blood, most of the triglyceride content of VLDLparticles is hydrolyzed by lipoprotein lipase, releasing free fattyacids to be used as a source of energy or for storage. As a result ofthis process, VLDL particles diminish in size and increase in densityand are converted into VLDL remnants or IDL. Further processing includesadditional lipolysis and exchange of lipids and apolipoproteins betweenIDL and HDL, leading to the formation of LDL which contain mostlycholesterol esters in the core and phospholipids and Apo B-100 on thesurface. LDL particles are taken up by the hepatic and extrahepaticcells via specific LDL-receptor.

The reverse cholesterol transport pathway starts with the secretion ofnascent HDL particles which are produced by the liver and intestine.These disk-like particles consist primarily of phospholipids surroundedby Apo A-I. They accept free cholesterol from peripheral tissues whichis esterified and trans-located into the core of HDL particles, whichbecome spherical and ready to deliver their cholesterol content tohepatocytes. During the degradation of VLDL and LDL, HDL particles alsoaccept free cholesterol and apolipoproteins from these lipoproteins.

Role of Lipoproteins in Atherosclerosis

Atherosclerosis is a chronic disease characterized by progressivedeposition of cholesterol, fibrous elements and minerals in arterialwalls. Atherosclerosis is the underlying pathophysiological process ofcoronary heart disease (CHD), one of the leading causes of death inWestern World (Report of the Working Group on Atherosclerosis of theNational Heart and Lung and Blood Institute, 2 (Washington, D.C.:Government Printing Office, 1981) DHEW Publication No. (NIH) 82-2035).Although development of CHD is a very complex process influenced by manycontributing factors, subintimal cholesterol deposition in coronaryarteries is one of the earliest and most important events during thecourse of disease. The major source of cholesterol found in arterialwall deposits is plasma lipoproteins. Because of their diverse metabolicroles and properties, lipoproteins associate differently with the riskof developing CHD.

LDL particles constitute approximately two-thirds of total cholesterol(TC) and form the primary atherogenic fraction of the serum cholesterol.Many epidemiological and clinical studies have shown that increased LDLlevels in the blood are associated with an increased risk of CHD. Forexample, the results of the Lipid Research Clinics trial have shown thatreduction of LDL-cholesterol (LDL-C) is associated with a significantdecrease in CHD incidence (The Lipid Research Clinics Coronary PrimaryPrevention Trial results:II. JAMA 251:365–374 (1984)).

The evidence relating CHD and triglyceride-rich lipoproteins such asVLDL is not as strong as for the LDL. Many studies have shown a positivecorrelation between elevated serum triglyceride levels and increasedrisk of CHD. However, the independent link between elevated serumtriglyceride (TG) and CHD breaks down when multivariate analyses areused to control statistically for the effects of total cholesterol (TC)and HDL-cholesterol (HDL-C). These observations suggest that increasedCHD risk noted in patients with hypertriglyceridemia could be due toeither the accumulation of triglyceride-rich particles that are uniquelyatherogenic in some people or to the association with reduced HDL-C(Assmann, G. et al., Am. J. Cardiol., 68:1–3 (1991)). Remnants oftriglyceride-rich particles, (for example, chylomicron and VLDLremnants) which are found in IDL are also atherogenic (Krauss, R. M.,Am. Heart J., 112:578–582 (1987)).

In contrast to the atherogenic potential of LDL, VLDL and VLDL remnants,HDL are inversely correlated with CHD, so that individuals with lowconcentrations of HDL-C have an increased incidence of CHD (Gordon, T.et al., Am. J. Med., 62:707–714 (1977); Miller, N. E. et al., Lancet,1:965–968 (1977); Miller, G. J. and Miller, N. E., Lancet, 1:16–19(1975)). At the other extreme, individuals with high concentrations ofHDL, such as found in familial hyperalphalipoproteinemia, seldom expresssymptoms of CHD. The fact that pre-menopausal females have higher HDLconcentrations and less CHD compared to males, also supports theanti-atherogenic role of HLD. Furthermore, postmenopausal women have asignificant increase in CHD risk while their HDL concentrationsdecrease.

Measurement of LDL

LDL consists of a hydrophobic lipid core composed of cholesterol estersand triglycerides. The lipid core of the LDL particle is surrounded byan amphipathic coat composed of phospholipids, unesterified cholesteroland Apo B. Each LDL particle contains one molecule of Apo B-100. On aweight basis, LDL is composed of 38 percent cholesterol ester, 22percent phospholipid, 21 percent protein, 11 percent triglyceride and 8percent unesterified cholesterol.

Accurate measurements of LDL using presently available technologydepends on separation of LDL particles from other lipoproteins. Once theLDL particles are separated, their concentration can be quantified bydetermination of their cholesterol (LDL-C) or Apo B (LDL-B) content.LDL-C is the most commonly used parameter.

Several ultracentrifugation methods have been developed over the yearsto separate serum lipoproteins. Analytical ultracentrifugation wasdeveloped in the 1950s and continues to be used today in some researchlaboratories. In this technique, lipoproteins are separated byanalytical ultra-centrifugation and quantitated by optical refraction.This method of quantitation measures lipoprotein mass, but does not giveany information about lipid or protein composition. Sequentialultracentrifugation was developed in 1955 to overcome some of thelimitations of analytical ultracentrifugation. In this technique,lipoproteins are separated by repeated ultracentrifugations afterprogressively increasing the sample density. Lipoproteins can beisolated within any desired density interval and in sufficientquantities to allow for multiple chemical analyses. Sequentialultracentrifugation continues to be used today for preparative isolationof lipoproteins. However, the ultracentrifugation methods are tooexpensive and time consuming for the purpose of measuring LDL-C levelsto assess lipoprotein abnormalities and CHD risk in routine clinicalapplication. Other methods for separating LDL include size-exclusion andother types of chromatography, electro-phoresis, and precipitation. Thesize-exclusion chromatography methods include agarose columnchromatography and high-performance gel filtration columnchromatography. The time required for analysis, typically 24 hours, isthe major difficulty with agarose column chromatography. The developmentof high-performance liquid chromatography (HPLC) methods has reduced theanalysis time, but has increased the cost and complexity of theprocedure. Affinity chromatography using anti-LDL antibodies, heparin,or dextran sulfate linked to SEPHAROSE™ (Pharmacia LKB, Piscataway,N.J.) gels has also been used to isolate LDL.

Electrophoresis methods, which separate lipoproteins according to theircharge in addition to size, have been used in many clinicallaboratories. This technique is helpful in qualitative assessment ofvarious types of hyperlipoproteinemias. Agarose gel electrophoresis atpH 8.6, followed by visualization using lipophilic stains such as OilRed O, Sudan Black B or Sudan Red 7B, have been commonly used withcommercial reagents packaged as kits, for example, as sold by CibaCorning (Medfield, Mass.) Lipoprotein concentrations are then estimatedby densitometry based on the color intensity of the separated bands.

Several methods for selective chemical precipitation of LDL have beendescribed and commercialized (Mulder, K. et al., Clin. Chim. Acta143:29–35 (1984)). The precipitation methods, which quantitate LDL-C asthe difference between the total cholesterol and the sum of VLDL- andHDL-cholesterol (Friedewald, W. T. et al., Clin. Chem. 18:499–502(1972)), are precise and produce reasonably accurate results relative toultracentrifugation methods when serum TG values are low. However, mostinvestigators have found that the precipitation methods are plagued withsystematic errors when samples with high TG levels are analyzed.

Most recently, a method was developed which uses latex beads coated withaffinity-purified polyclonal goat antisera directed againstapolipoproteins in HDL and VLDL (Sigma, St. Louis, Mo.). In this method,a plasma or serum sample is incubated with the beads for 5 to 10 minutesat room temperature and then centrifuged for 5 minutes to remove the HDLand VLDL bound to the beads. The remainder of the sample is then assayedfor cholesterol using a standard enzymatic cholesterol assay (Sigma St.Louis, Mo.) to obtain a value for the LDL-C, the presumed remainingsource of cholesterol in the sample.

Techniques used for measurement of LDL by its Apo B content includeradioimmunoassay; enzyme immunoassay (ELISA competitive or capturesystems), fluorescence immunoassay, radial immunodiffusion,nephelometry, turbidimetry and electroimmunoassay.

The National Cholesterol Education Program (NCEP) recommended thedetermination of LDL-C concentration in diagnosis and treatment ofhypercholesterolemia. According to NCEP, concentrations lower than 130mg/dl in adults are considered desirable, concentrations between 130 and150 mg/dl are borderline high, and concentrations above 160 md/dl arehigh (see Report of the National Cholesterol Education Program. ExpertPanel on Detection, Evaluation and Treatment of High Blood Cholesterolin Adults, Arch. Intern. Med. 148:36–69 (1988)). The NCEP alsorecommended determining LDL-cholesterol concentrations for children andadolescents since the high LDL correlates with the extent of coronaryand aortic atherosclerosis in this age group as well as development ofCHD later in life. Cholesterol values of 110 mg/dl are desirable, valuesbetween 110 and 129 mg/dl are borderline high, and values above 130mg/dl are considered high in children and adolescents (see Report of theExpert Panel on Blood Cholesterol Levels in Children and Adolescents,Pediatrics 89:525–584 (1992)).

Measurement of HDL

HDL, the smallest in size of the lipoproteins, includes a family oflipoprotein particles that exist in a constant state of dynamic flux asthey interact with LDL, IDL and VLDL. HDL have the highest proportion ofprotein (50 percent) relative to lipid compared to other lipoproteins.The major HDL proteins are Apo A-I and Apo A-II, with lowerconcentrations of Apo C (I, II & III), E, and A-IV. Phospholipids arethe principal lipid component of HDL, with cholesterol esters,unesterified cholesterol, and TG present in lower concentrations.

As in the case of LDL, HDL is typically measured after its separationfrom other lipoproteins and quantification of cholesterol in the HDL(HDL-C). As described above, the separation of HDL can be accomplishedby ultracentrifugation, chromatographic procedures, electrophoresis andprecipitation. The reliability of lipoprotein quantitations followingseparation by ultracentrifugation techniques depends upon both theperformance of the analytical quantitation method, such as cholesterolanalysis, and the skills of the technologist in performing accuraterecovery and transfer of the lipoprotein fractions from theultracentrifuge tube. HDL-C is more easily quantitated by selectiveprecipitation techniques compared to either ultracentrifugation orelectrophoretic methods. Currently, the majority of clinicallaboratories use either dextran sulfate or sodium phosphotungstateprocedures for HDL-C analysis (Warnick, G. R. et al., Clin. Chem.28:1379–1388 (1982); Lopes-Virella, M. F. et al., Clin. Chem. 23:882–884(1977)). According to NECP guidelines, patients with HDL-C levels below35 mg/dl are considered to be at risk for CHD.

LDL/HDL Ratio

Some studies have demonstrated that the ratio between LDL-C and HDL-Crepresents a better predictor of CHD than either of these two parametersalone (Arntzenius, A. C., Acta. Cardiol., 46:431–438 (1991); Barth J. D.and Arntzenius, A. C., Eur. Heart J., 12:952–957 (1991); Ortola, J. etal., Clin. Chem., 38:56–59 (1992); Gohlke H., Wien Klin. Wochenschr.,104:309–313 (1992)).

LPA-I and LPA-I:A-II Lipoprotein Particles

There are two subpopulations of HDL lipoprotein particles known as LPA-Iand LPA-I:A-II (Koren, E. et al. Clin. Chem., 33:38–43 (1987)). LPA-Iparticles contain Apo A-I but no Apo A-II while LPA-I:A-II particlescontain both apolipoproteins. These HDL subpopulations can be measuredby enzyme immunoassay (Koren, E. et al. Clin. Chem., 33:38–43 (1987)) orelectroimmunoassay (Atmeh, R. F. et al., Biochim. Biophys. Acta,751:175–188 (1983)). Their importance has been emphasized by severalstudies which demonstrated that LPA-I is a more active component inreverse cholesterol transport and, there-fore, more anti-atherogenicthan other lipoproteins (Puchois, P. et al., Atherosclerosis, 68:35–40(1987); Fruchart, J. C. and Ailhaud, G., Clin. Chem., 38:793–797(1992)).

Measurements of VLDL, IDL, C-III and E ratios

Triglyceride-rich VLDL as well as their remnants (IDL) can be separatedby the above ultracentrifugational, chromatographic and electrophoreticmethods and quantified by determination of their cholesterol content.Although atherogenic, these lipoprotein particles are not commonlymeasured in routine clinical laboratories. Instead, serum triglycerideconcentration in the fasting state is considered representative of theVLDL content and is used traditionally in the assessment of theVLDL-related CHD risk. More recently, measurements of the so-calledC-III and E ratios have been proposed as reliable predictors of theVLDL-related CHD risk. The principle of these measurements is toprecipitate all Apo B-100-containing particles (VLDL, IDL and LDL) withheparin which leaves HDL in the heparin supernate. This separation isfollowed by an immunochemical determination of Apo C-III or Apo E in theheparin precipitate and heparin supernate and calculation of thecorresponding ratios by dividing C-III or E concentration in heparinsupernate with their respective concentrations in heparin precipitate(Alaupovic, P., Can. J. Biochem., 59:565–579 (1981)). Most of the ApoC-III and Apo E in the heparin precipitate is associated with Apo B inVLDL and VLDL remnant (IDL) particles. The C-III and E in the heparinsupernate is associated with Apo A-I in HDL particles. Apo C-III and/orApo E in the heparin precipitate reflects the concentration of VLDL andVLDL-remnant particles both of which are atherogenic. The Apo C-III andApo E in the heparin supernate represents HDL particles which areanti-atherogenic. Therefore, a low C-III and E ratio is associated withincreased risk of CHD because it reflects either high VLDL and IDL andnormal HDL or, more frequently, high VLDL and IDL combined with low HDL.In fact, the predictive power of C-III ratio has surpassed that oftriglycerides in several clinical studies (Alaupovic, P. andBlankenhorn, D. H., Klin. Wochenschr., 60:38–40 (1990); Blankenhorn, D.H. et al., Circulation 81:470–478 (1990)).

Measurements of Apo A-I and B

Apo B-100 is an integral component of the four major atherogeniclipoproteins: VLDL, IDL, LDL and Lp(a). Apo B-100 is distinguished fromApo B-48, which is found only in lipoproteins of intestinal origin, suchas chylomicrons and chylomicron remnants. Apo B-48 is usuallyundetectable in the systemic circulation, except in rare subjects withType I, III, or V hyperlipidemia. Apo B's initial function in VLDL andIDL appears to be structural; however, with exposure of binding domainson LDL, it becomes responsible for interaction with high-affinity LDLreceptors on cell surfaces, which results in uptake and removal of LDLfrom the circulation. Several studies have shown that an increased Apo Blevel in blood is a reliable marker for coronary atherosclerosis(Sniderman, A. et al., Proc. Natl. Acad. Sci. USA, 77:604–608 (1980);Kwiterovich, P. O. et al., Am. J. Cardiol., 71:631–639 (1993); McGill etal. Coron. Artery Dis., 4:261–270 (1993); Tornvall, P. et al.,Circulation, 88:2180–2189 (1993)).

Apo A-I is the major protein constituent of lipoproteins in the highdensity range. Apo A-I may also be the ligand that binds to a proposedhepatic receptor for HDL removal. A number of studies support theclinical sensitivity and specificity of Apo A-I as a negative riskfactor for atherosclerosis (Avogaro, P. et al., Lancet, 1:901–903(1979); Maciejko, J. J. et al., N. Engl. J. Med., 309:385–389 (1983)).Some investigators have also described Apo A-I/Apo B ratio as a usefulindex of atherosclerotic risk (Kwiterovich, P. O. et al., Am. J.Cardiol., 69:1015–1021 (1992); Kuyl, J. M. and Mendelsohn, D., Clin.Biochem., 25:313–316 (1992)).

Techniques used for both Apo A-I and B are confined to immunologicalprocedures using antibodies directed against Apo A-I or B and includeradio-immunoassay (RIA), enzyme immunoassay (ELISA), competitive orcapture systems, fluorescence immunoassay, radial immunodiffusion,nephelometry, turbidimetry and electroimmunoassay.

To summarize, there are several lipoprotein related parameters that arecurrently used as predictors of CHD. Some of them represent atherogeniclipoproteins (total cholesterol, triglycerides, LDL, IDL, VLDL, Lp(a)and Apo B and are positively associated with CHD whereas the others areanti-atherogenic factors, HDL, Apo A-I and LPA-I which are inverselyrelated to the disease. The ratios of some of these parameters, such asLDL/HDL, Apo A-I/Apo B, C-III and E ratio, appear to be even moresensitive predictors of CHD because each of them reflects bothanti-atherogenic and atherogenic factors in a single parameter.

All of the methods currently used to determine lipoprotein related riskfactors require a laboratory with the necessary equipment and trainedpersonnel to carry out each of the technical steps, to perform thenecessary calculations and to interpret the results. The only exceptionis a new total cholesterol measurement device (AccuMeter CholesterolSelf-Test) developed by ChemTrack (Sunnyvale, Calif.) and designed forhome use. However, a total cholesterol level is a less sensitivepredictor compared to the levels of specific lipoproteins,apolipoproteins or ratios thereof.

It is therefore an object of the present invention to provide methodsand means to rapidly and reliably determine levels of specificlipoproteins, apolipoproteins or the ratios thereof in whole blood,serum or plasma without the necessity of laboratory equipment ortechnically trained personnel.

It is another object of the present invention to provide antibodiesimmunoreactive with specific epitopes on lipoproteins, such as those onLDL, VLDL and HDL, that enable rapid and reliable determinations oflevels of lipoproteins and/or apolipoproteins in whole blood, serum orplasma.

SUMMARY OF THE INVENTION

Compositions and methods using antibodies which are immunoreactive withspecific apolipoproteins to determine the concentrations of lipoproteinssuch as HDL and LDL, and/or apolipoproteins in human blood, serum orplasma sample, are described. Monoclonal antibodies (MAbs) are describedthat specifically bind to epitopes present in apolipoproteins andlipoproteins, enabling rapid and reliable determinations of levels ofspecific blood lipoprotein and/or apolipoprotein levels, including ApoB-100, Apo A-I, Apo A-II, Apo C-III, and Apo E, and therebydetermination of relative ratios of HDL and LDL and LpaI and LpaII. In apreferred embodiment, the compositions are strips of a solid phasematerial coated with one or more of the antibodies and are referred toherein as “dipsticks”. The dipsticks specifically bind a lipoprotein orapolipoprotein when dipped into a protein sample. The amount of lipidassociated with a bound lipoprotein or the amount of apolipoproteinbound on the dipstick is quantitated using an appropriate method, forexample, by staining with a lipid stain or reaction with a secondlabelled antibody. The intensity of the stain on the dipstick isproportional to the concentration of the lipoprotein lipid orapolipoprotein circulating in the blood and can be quantitated bycomparison with standards containing known amounts of lipid. Thedipsticks can be provided alone or in kits which enable the lay personto carry out the assay without the need of a physician or technicallaboratory.

The MAbs can be used not only as components of dipsticks, but also in avariety of other methods, including enzyme immunoassays,radioimmunoassays as well as fluorescent and chemiluminescentimmunoassays to determine lipoproteins and apolipoproteins in biologicalsamples, and in purification of the apolipoprotein or lipoprotein withwhich they are immunoreactive.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions to determine the concentration of specificlipoproteins and/or apolipoproteins, such as LDL and HDL, which whenpresent at elevated levels in the body are causally related to anincreased or decreased risk of CHD have been developed. In the preferredembodiment, blood lipoprotein and/or apolipoprotein molecules in apatient sample are bound to specific antibodies immobilized on speciallyprepared strips of solid phase material and to the bound lipoproteinand/or apolipoproteins visualized using specific colored stainingreagents. The intensity of the color is proportional to theconcentration of the lipid component or apolipoprotein component of thelipoprotein circulating in the blood.

I. Antibodies to Lipoproteins and Apolipoproteins

A. MAb Methodology

Monoclonal antibody technology can be used to obtain MAbs useful inmethods to rapidly and reliably determine blood lipoproteins andapolipoproteins (Galfré, G. and Milstein, C., Methods Enzymol., 73:3–46(1981) incorporated herein by reference). Briefly, hybridomas areproduced using spleen cells from mice immunized with a particularapolipoprotein. The spleen cells of each immunized mouse is fused withmouse myeloma Sp 2/0 cells, for example using the polyethylene glycolfusion method of Galfré, G. and Milstein, C., Methods Enzymol., 73:3–46(1981). Growth of hybridomas, selection in HAT medium, cloning andscreening of clones against antigens are carried out using standardmethodology (Galfré, G. and Milstein, C., Methods Enzymol., 73:3–46(1981)).

HAT-selected clones are injected into mice to produce large quantitiesof MAb in ascites as described by Galfré, G. and Milstein, C., MethodsEnzymol., 73:3–46 (1981), which can be purified using protein A columnchromatography (BioRad, Hercules, Calif.). MAbs are selected on thebasis of their (a) specificity for a particular apolipoprotein, (b) highbinding affinity, (c) isotype, and (d) stability.

B. Testing for Specificity and Affinity

MAbs can be screened or tested for specificity using any of a variety ofstandard techniques, including Western Blotting (Koren, E. et al.,Biochim. Biophys. Acta 876:91–100 (1986)) and enzyme-linkedimmunosorbent assay (ELISA) (Koren, E. et al., Biochim. Biophys. Acta876:91–100 (1986)), as described in more detail in the followingexamples.

In ELISA, separate wells in microtiter plates are coated with purifiedapolipoproteins which adsorb to the wall of the wells. The wells arethen treated with a blocking agent, such as bovine serum albumin ornonfat milk proteins, to cover areas in the wells not bound by antigen.Ascites fluid or other antibody-containing preparation can then beapplied to each well in varying concentrations and adequate time allowedfor MAb to bind the antigen adsorbed on the wall of each well. Thepresence of MAb bound to antigen in a well can then be detected using astandard enzyme-conjugated anti-mouse antibody which will bind MAb thathas bound to apolipoprotein in the well. Wells in which MAb is bound toantigen are then identified by adding a chromogenic substrate for theenzyme conjugated to the anti-mouse antibody and color productiondetected by an optical device such as an ELISA plate reader.

MAbs that bind to a single apolipoprotein with no significant detectablecrossreactivity with other apolipoproteins are considered specific. Todetermine specificity of MAbs for a particular lipoprotein, individualwells on ELISA plates are coated with purified chylomicrons VLDL, LDLand HDL and subjected to the identical procedure. To determine whetheror not two MAbs specific for the same apolipoprotein bind to differentepitopes, a competitive ELISA is performed. For example, one of the MAbsis biotinylated. Mixtures containing a constant concentration of thebiotinylated MAb and increasing concentrations of the nonbiotinylatedMAb are incubated with wells coated with the apolipoprotein orlipoprotein antigen. Quantity of biotinylated antibody bound to thecoated antigen is determined using a streptavidin-peroxidase conjugateand a chromogenic substrate. Decreased binding of the biotinylated MAbwith increasing concentrations of the nonbiotinylated MAb indicates thatthe two MAbs compete for the same epitope. If the biotinylated MAb bindsequally to the antigen as does the unlabelled MAb despite increasingconcentrations of the nonbiotinylated MAb, the two antibodies do notcompete for the same epitope. This competition can be complete orpartial. Affinity of MAbs can be determined using radioactively labelled(¹²⁵I) lipoproteins or apolipoproteins and purified MAbs as described byKoren, E. et al., Biochim. Biophys., Acta 876:91–100 (1986),incorporated herein by reference).

C. Proteolytic Cleavage of Antibodies

It may be desirable to produce and use functional fragments of an MAbfor a particular application. The well-known basic structure of atypical IgG molecule is a symmetrical tetrameric Y-shaped molecule ofapproximately 150,000 to 200,000 daltons consisting of two identicallight polypeptide chains (containing about 220 amino acids) and twoidentical heavy polypeptide chains (containing about 440 amino acids).Heavy chains are linked to one another through at least one disulfidebond. Each light chain is linked to a contiguous heavy chain by adisulfide linkage. An antigen-binding site or domain is located in eacharm of the Y-shaped antibody molecule and is formed between the aminoterminal regions of each pair of disulfide linked light and heavychains. These amino terminal regions of the light and heavy chainsconsist of approximately their first 110 amino terminal amino acids andare known as the variable regions of the light and heavy chains. Inaddition, within the variable regions of the light and heavy chainsthere are hypervariable regions which contain stretches of amino acidsequences, known as complementarity determining regions (CDRs). CDRs areresponsible for the antibody's specificity for one particular site on anantigen molecule called an epitope. Thus, the typical IgG molecule isdivalent in that it can bind two antigen molecules because eachantigen-binding site is able to bind the specific epitope of eachantigen molecule. The carboxy terminal regions of light and heavy chainsare similar or identical to those of other antibody molecules and arecalled constant regions. The amino acid sequence of the constant regionof the heavy chains of a particular antibody defines what class ofantibody it is, for example, IgG, IgD, IgE, IgA or IgM. Some classes ofantibodies contain two or more identical antibodies associated with eachother in multivalent antigen-binding arrangements.

Proteolytic cleavage of a typical IgG molecule with papain is known toproduce two separate antigen binding fragments called Fab fragmentswhich contain an intact light chain linked to an amino terminal portionof the contiguous heavy chain via by disulfide linkage. The remainingportion of the papain-digested immunoglobin molecule is known as the Fcfragment and consists of the carboxy terminal portions of the antibodyleft intact and linked together via disulfide bonds. If an antibody isdigested with pepsin, a fragment known as an F(ab′)₂ fragment isproduced which lacks the Fc region but contains both antigen-bindingdomains held together by disulfide bonds between contiguous light andheavy chains (as Fab fragments) and also disulfide linkages between theremaining portions of the contiguous heavy chains (Handbook ofExperimental Immunology, Vol 1: Immunochemistry, Weir, D. M., Editor,Blackwell Scientific Publications, Oxford (1986)).

Fab and F(ab′)₂ fragments of MAbs that bind particular bloodapolipoproteins or lipoproteins can be used in place of whole MAbs inmethods for detecting or quantifying such blood proteins or the lipidsassociated with such proteins. Because Fab and F(ab′)₂ fragments aresmaller than intact antibody molecules, more antigen-binding domains canbe immobilized per unit area of a solid support than when whole antibodymolecules are used. As explained below, rapid, easy and reliable assaysystems can be made in which antibodies or antibody fragment thatspecifically bind apolipoproteins and lipoproteins are immobilized onsolid phase materials.

D. Recombinant Antibodies

Recombinant DNA methods have been developed which permit the productionand selection of recombinant antibodies which are single chainantigen-binding polypeptides known as single chain Fv fragments (ScFvsor ScFv antibodies). ScFvs bind a specific epitope of interest and canbe produced using any of a variety of recombinant bacterial phage-basedmethods, for example as described in Lowman, H. B. et al., Biochemistry,30: 10832–10838 (1991); Clackson, T. et al., Nature, 352: 624–628(1991); and Cwirla, S. E. et al., Proc. Natl. Acad. Sci. USA, 87:6378–6382 (1990), incorporated herein by reference. These methods areusually based on producing genetically altered filamentous phage, suchas recombinant M13 or fd phages, which display on the surface of thephage particle a recombinant fusion protein containing theantigen-binding ScFv antibody as the amino terminal region of the fusionprotein and the minor phage coat protein g3p as the carboxy terminalregion of the fusion protein. Such recombinant phages can be readilygrown and isolated using well-known phage methods. Furthermore, theintact phage particles can usually be screened directly for the presence(display) of an antigen-binding ScFv on their surface without thenecessity of isolating the ScFv away from the phage particle.

To produce an ScFv, standard reverse transcriptase protocols are used tofirst produce cDNA from mRNA isolated from a hybridoma that produces anMAb for an antigen of interest. The cDNA molecules encoding the variableregions of the heavy and light chains of the MAb can then be amplifiedby standard polymerase chain reaction (PCR) methodology using a set ofprimers for mouse immunoglobulin heavy and light variable regions(Clackson, T. et al., Nature, 352:624–628 (1991), incorporated herein byreference). The amplified cDNAs encoding MAb heavy and light chainvariable regions are then linked together with a linker oligonucleotidein order to generate a recombinant ScFv DNA molecule. The ScFv DNA isligated into a filamentous phage plasmid designed to fuse the amplifiedcDNA sequences into the 5′ region of the phage gene encoding the minorcoat protein called g3p. Escherichia coli bacterial cells are thantransformed with the recombinant phage plasmids, and filamentous phagegrown and harvested. The desired recombinant phages displayantigen-binding domains fused to the amino terminal region of the minorcoat protein. Such “display phages” can then be passed over immobilizedantigen, for example, using the method known as “panning”, see Parmley,S. F. and Smith, G. P., Adv. Exp. Med. Biol., 251:215–218 (1989);Cwirla, S. E. et al., Proc. Natl. Acad. Sci. USA, 87: 6378–6382 (1990),incorporated herein by reference, to adsorb those phage particlescontaining ScFv antibody proteins that are capable of binding antigen.The antigen-binding phage particles can then be amplified by standardphage infection methods, and the amplified recombinant phage populationagain selected for antigen-binding ability. Such successive rounds ofselection for antigen-binding ability, followed by amplification, selectfor enhanced antigen-binding ability in the ScFvs displayed onrecombinant phages. Selection for increased antigen-binding ability maybe made by adjusting the conditions under which binding takes place torequire a tighter binding activity. Another method to select forenhanced antigen-binding activity is to alter nucleotide sequenceswithin the cDNA encoding the binding domain of the ScFv and subjectrecombinant phage populations to successive rounds of selection forantigen-binding activity and amplification (see, Lowman, H. B. et al.,Biochemistry, 30: 10832–10838 (1991) and Cwirla, S. E. et al., Proc.Natl. Acad. Sci. USA, 87: 6378–6382 (1990)).

Once an ScFv is selected, the recombinant antibody can be produced in afree form using an appropriate vector in conjunction with E. coli strainHB2151. These bacteria actually secrete ScFv in a soluble form, free ofphage components (Hoogenboom H. R. et al., Nucl. Acids Res.,19:4133–4137 (1991), incorporated herein by reference). The purificationof soluble ScFv from the HB2151 bacteria culture medium can beaccomplished by affinity chromatography using antigen moleculesimmobilized on a solid support such as AFFIGEL™ (BioRad, Hercules,Calif.).

More recent developments in the recombinant antibody technologydemonstrate possibilities for further improvements such as increasedavidity of binding by polymerization of ScFvs into dimers and tetramers(Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90: 6444–6448 (1993);Mezes, P. Construction and Biodistribution Studies of MultivalentSingle-Chain Antibodies, The Fourth Annual IBC International Conferenceon Antibody Engineering, December 1993, Coronado, Calif.,; Ito, W. andKurosawa, Y., J. Biol. Chem., 268: 20668–20675 (1993), incorporatedherein by reference).

Because ScFvs are even smaller molecules than Fab or F(ab′)₂ fragments,they can be used to attain even higher densities of antigen bindingsites per unit of surface area when immobilized on a solid supportmaterial than possible using whole antibodies, F(ab′)₂, or Fabfragments. Furthermore, recombinant antibody technology offers a morestable genetic source of antibodies, as compared with hybridomas.Recombinant antibodies can also be produced more quickly andeconomically using standard bacterial phage production methods.

As demonstrated below, the availability of hybridomas which produce MAbsto Apo B-100, Apo A-I, Apo A-II, Apo C-III, and Apo E enables theproduction of recombinant antibodies to these same antigens.

E. Anti-Apo Monoclonal Antibodies (MAbs)

Unless specifically stated otherwise, the term “MAbs” includes naturaland recombinant antibodies and fragments thereof.

MAbs to apolipoprotein (Apo) A-I, A-II, B, C-III and E can be usedeither alone or in various combinations to obtain a useful determinationof the body's circulating levels of lipoproteins and/or apolipoproteins.MAbs used for making dipsticks, such as HB₃cB₃, D₆, AIbD₅, and CdB₅,described below, possess very high affinity constants ranging from 10⁹to 10¹² M⁻¹ as determined by the methods described by Koren, et al.,Biochim. Biophys. Acta, 876:91–100 (1986); Biochim. Biophys. Acta,876:101–107 (1986), incorporated herein by reference. An antibodycoating a solid phase material is expected to bind a sufficient quantityof lipoprotein within a relatively short period of time (approximatelytwo to five minutes), and to retain the captured lipoprotein duringsubsequent washing and staining for bound lipoprotein. It should beunderstood that while the descriptions below are the best antibodiespresently known for making the compositions described herein, themethods described or incorporated by reference herein by citation toprior publications can be used by those skilled in the art to make othersuitable antibodies having similar affinity and specificity which arefunctionally equivalent to those used in the following examples.

Monoclonal antibodies (MAbs) to apolipoproteins A-I, A-II, C-III and Ewere produced by immunization of Balb/c mice (Jackson Laboratories, St.Louis, Mo.) with purified apolipoproteins. All apolipoproteins werepurified using well-established methods (Curry, M. D. et al., Clin.Chem. 22:315–322 (1976); Curry, M. D. et al., Clin. Chem. 24:280–286(1978); Curry, M. D. et al., Biochim. Biophys. Acta 439:413–425 (1976);and Curry, M. D. et al., Biochim. Biophys. Acta 617:503–513 (1980)).

From a library of several hundred MAbs, two antibodies directed againstApo A-I, one against Apo A-II, two against Apo B, one against Apo C-IIIand two against Apo E were selected for the methods and compositionsdescribed herein. The MAbs were selected on the basis of their (a)specificity, (b) high binding affinity, (c) isotype (class of antibody),and (d) stability under the conditions described below. Usingcommercially available isotype specific anti-mouse antibodies(Kirkegaard and Perry Laboratory, Gaithersburg, Md.) all of the MAbswere shown to belong to the IgG1 class and possess kappa light chains.

Antibodies to Apo B

Two MAbs specific for Apo B, D₆ and HB₃cB₃ MAbs, were developed andfound to be useful for the methods and compositions described below. D₆and HB₃cB₃ MAbs bind to sterically distant epitopes on Apo B.

Antibodies to Pan B

D₆ MAb is an antibody with equal binding and high affinity for all ApoB-containing lipoproteins in human plasma, as described by Koren, E. etal., Biochim. Biophys. Acta, 876:91–100 (1986); Koren, E. et al.,Biochim. Biophys. Acta, 876:101–107 (1986), specifically including ApoB-48 and Apo B-100. D₆ binds to an epitope localized at the aminoterminal half of Apo B and recognizes both B-48 and B-100.

D₆ was produced after immunization of mice with a narrow cut of lowdensity (1.021 to 1.006 g/ml) lipoproteins (LDL) containingapolipoprotein B (Apo B) as a sole protein (Smith, L. C. et al., Ann.Rev. Biochem., 47: 751–777 (1978)). Hybridomas were produced usingspleen cells from immunized mice. The fusion of spleen cells with mousemyeloma Sp 2/0 cells was carried out using the polyethylene glycolmethod of Galfré, G. and Milstein C., Methods Enzymol., 73: 3–46 (1981).Growth of hybridomas, selection in HAT medium, cloning and screening ofhydridoma clones against specific antigens were carried out usingstandard methodology (Galfré, G. and Milstein C., Methods Enzymol., 73:3–46 (1981), incorporated herein by reference). Selected clones wereinjected into mice to produce large quantities of antibodies in ascites(Galfré, G. and Milstein C., Methods Enzymol., 73: 3–46 (1981)) followedby purification of MAbs using protein A column chromatography (Bio-Rad,Hercules, Calif.).

Antibodies to Apo B-100

Conventional ways of producing MAbs to Apo B-100 include immunization ofmice with LDL. This approach is convenient because it is relativelysimple to isolate LDL. However, MAbs produced using LDL as an immunogentend to be sensitive to conformational changes of Apo B-100 caused byvariations in the lipid composition of LDL particles. For example, ApoB-100 epitopes are less reactive with a number of anti-Apo B MAbs due tothe presence of various amounts of triglycerides (Keidar, S. et al.,Metabolism, 39: 281–288 (1990); Galeano, N. F. et al., J. Biol. Chem.,269:511–519 (1994); Harduin, P. et al., Arterioscl. Thromb., 13: 529–535(1993)).

For the methods and compositions described herein, an MAb is desiredthat fulfills two important criteria: (i) selective recognition of LDLand (ii) high and invariable reactivity with LDL particles, irrespectiveof possible variations in their lipid composition and/or conformation.Such an MAb must, therefore, recognize a stable,conformation-independent epitope which is uninfluenced by the lipidcontent and which is equally expressed in all LDL particles, butinaccessible in VLDL and chylomicrons. A MAb possessing these propertieshas not been previously described. For example, a detailed comparison oftwo known, potentially LDL specific MAbs demonstrated that neither ofthem can meet the above requirements (Milne, R. et al., J. Biol. Chem.,264:19754–19760 (1989); WO 93/18067). Cross-reactivity with VLDL,especially in samples with high VLDL concentrations appears to be themajor obstacle even in the case of most promising “anti-LDL” MAbs suchas 8A2.1 and 4B5.6 (WO93/18067) (La Belle, M. et al., Clin. Chim. Acta,191:153–160 (1990)). To obtain an anti-LDL MAb whose binding to LDLparticles is not dependent on variations in LDL composition and/orconformation, mice were immunized with soluble Apo B-100 which had beendelipidized, reduced, carboxymethylated and, purified byelectrophoration in polyacrylamide gels containing 8 M urea (Lee, D. M.et al., Biochim. Biophys. Acta, 666: 133–146 (1981)). Immunization withsuch delipidized, soluble, reduced, carboxymethylated, andelectrophoretically purified Apo B-100 has not been previously reported.

The spleen cells of mice that were immunized using the soluble andelectrophoretically purified Apo B, were then used to produce hybridomasaccording to standard hybridoma methods. A resulting MAb, HB₃cB₃, bindsselectively to LDL particles produced by a hybridoma generated usingspleen cells immunized with the soluble and electrophoretically purifiedApo B.

HB₃cB₃ binds to the epitope near the T2 carboxy terminal region ofB-100, exclusively, and does not recognize B-48. The epitope recognizedby HB₃cB₃ may be conformationally changed or masked by lipids and/orother apolipoproteins present in VLDL. Chylomicrons are not recognizedby HB₃cB₃ because they lack Apo B-100. The HB₃cB₃ MAb, and LDL-bindingfragments derived therefrom, can be used as an LDL-specific bindingmolecule in all of the compositions and methods described herein becauseof its specificity for LDL and lack of cross-reactivity with otherlipoproteins.

Antibodies to Apo A-I

Two MAbs raised against apolipoprotein A-I were selected from a libraryof MAbs for developing rapid and sensitive means and methods ofdetecting lipoproteins predictive of risk of CHD. Both of them bind toHDL with a high affinity and show negligible reactivity with any otherlipoprotein density class. The two anti-Apo A-I MAbs, AIbD₅ and AIbE₂,bind to sterically distant epitopes since they do not compete with eachother in their binding to either delipidized and purified Apo A-I orintact HDL particles. Both MAbs to Apo A-I bind with high affinity todelipidized Apo A-I and to HDL and show negligible or no binding to LDL,VLDL, chylomicrons and Apos A-II, C-III and E as shown in Tables 1 and 2below.

Antibodies to Apo A-II

An MAb to Apo A-II was produced using purified Apo A-II as an immunogen.This antibody binds with high affinity to HDL and is capable of removingall the HDL particles containing Apo A-II (LP-A-I:A-II particles) fromplasma or serum, leaving the HDL particles without Apo A-II (LP-A-Iparticles) intact. This anti-Apo A-II MAb, CdB₅, is described by Koren,E. et al., Arteriosclerosis, 6:521a (1986); Alaupovic, P. et al., J.Lipid Res., 32:9–19 (1991).

Antibodies to Apo C-III

An MAb to Apo C-III, XbA₃, which is useful in quantification of VLDLparticles is described by Koren, E. et al., Atherosclerosis, 95:157–170(1992).

Antibodies to Apo E

Two MAbs to Apo E are described by Koren, E. et al., Atherosclerosis,95:157–170 (1992). One of them, EfB₁, binds preferably to Apo Eassociated with VLDL which are precipitated by heparin whereas the other(EfD₃) binds predominantly to Apo E in HDL which are not precipitated byheparin treatment of a sample.

II. MAbs Immobilized on Solid Phase Materials.

A. Dipsticks

Antibodies can be bound to a solid phase material for use in assays orpurification procedures described herein. Various types of adsorptivematerials, such as nitrocellulose, Immobilon™ , polyvinyldienedifluoride (all from BioRad, Hercules, Calif.) can be used as a solidphase material to bind the anti-lipoprotein antibodies. Other solidphase materials, including resins and well-plates or other materialsmade of polystyrene, polypropylene or other synthetic polymericmaterials can also be used. In the preferred embodiment for assayinglipoprotein concentrations, pieces or strips of these materials arecoated with one or more antibodies, or functional fragments thereof,directed against specific epitopes of HDL, LDL, other lipoproteins, orapolipoproteins for use in patient samples. Such strips are referred toherein as “dipsticks”. The dipsticks may also be attached to one end ofa longer strip of a solid support material, such as plastic, which canserve as a handle for dipping a dipstick into a solution or sample, suchas a sample of whole blood, blood plasma, or blood serum. The plastichandle can also serve as a tether so that multiple dipsticks can beattached to a common support. Such a multi-strip design may beparticularly useful in a set-up for testing multiple lipoproteins and/orapolipoproteins simultaneously.

Although various sizes of dipsticks are possible, typically, pieces ofthe solid phase material that are coated with antibody have the generaldimensions of 0.5 cm×0.5 cm and can be attached to the longer solidsupport strips having general dimensions of 0.5 cm×5 cm. Such dimensionspermit an accurate determination of lipoprotein or apolipoprotein levelsin as little as 100 μl of blood.

The dipsticks described herein contain one or more regions containingimmobilized antibodies specific for particular epitopes onapolipoproteins or lipoproteins.

B. Coating Solid Phase Material with Antibodies

Adsorption

The strips of solid phase material, as used to make dipsticks, may becoated with antibodies by any of a variety of methods. If the strips aremade of a protein-receptive solid phase material that adsorbsantibodies, such as nitrocellulose or polyvinyldiene difluoride (PVDF)membrane, the material can be coated directly with antibody by immersingthe solid phase material directly into a solution of antibody. However,a random interaction between the antibody molecules and the solid phasematerial can occur with this method and a certain percentage (up to 30percent) of the antibody molecules that adsorb to the strips areimmobilized in an orientation that makes their antigen-binding sitesunavailable to bind their cognate lipoprotein or apolipoprotein antigenmolecules.

Avidin-Biotin Complexes

The proportion of antibody molecules on the dipsticks which arecorrectly oriented to bind their cognate antigens can be substantiallyincreased if antibody molecules are attached to the solid phase materialusing avidin-biotin complexes. The strips of solid phase material arefirst coated with avidin or streptavidin (both available commercially,for example, from Sigma Chemical Co., St. Louis, Mo.). PVDF strips canbe coated with avidin by incubating the strips in a solution of avidin(10 mg avidin in 3.5 ml of phosphate buffered saline, PBS) for 48 hoursat 4° C. The avidin-coated strips are then incubated with antibodymolecules which were previously biotinylated in their Fc domains (forexample, using a biotin-LC-hydrazide labelling kit, Pierce, Rockford,Ill.). The avidin molecules adsorbed on the solid phase materialspecifically bind the biotin linked to the Fc domains of the antibodies.In this way, the antibodies become attached to the solid phase material,optimally oriented with their carboxy terminal Fc regions linked to thesurface of the dipstick (via numerous biotin-avidin complexes) and withtheir antigen-binding domains directed away from the surface of thedipsticks and available for binding their cognate lipoprotein orapolipoprotein antigens in solution. Alternatively, the same linkage canbe achieved by chemically coupling biotin to the solid phase materialand covalently attaching avidin to the Fc portion of the antibodymolecules.

Use of the avidin (or streptavidin)-biotin system to coat strips withantibody yields dipsticks with a significantly higher capacity forbinding lipoproteins and/or apolipoproteins than dipsticks which wasmade by simply applying antibody directly to the strips of solid phasematerial. The higher binding capacity of the dipsticks containingantibodies adsorbed to the solid phase using the avidin-biotinconjugation system results in a more sensitive dipstick. This is of aparticular importance when dipsticks pre-stained with lipid or proteinstains are used to capture lipoproteins, as described below.

After antibody has been adsorbed directly on the protein receptivedipstick material, or indirectly through avidin (or streptavidin), thestrips are treated (“blocked”) with a blocking agent in order tominimize nonspecific adsorption of lipoproteins, lipids, orapolipoproteins to unoccupied sites on the dipstick material. Thedipsticks are treated with any of a variety of blocking agents such asbovine serum albumin (BSA), gelatin, Tris™, all of which are availablecommercially (Sigma, St Louis, Mo.) or nonfat milk proteins. Forexample, avidin-coated PVDF strips can be blocked with 2 percent (w/v)milk blocking solution (Kirkegaard and Perry Laboratories, Gaithersburg,Md.) for 48 hours at 4° C.

Antibodies can also be chemically coupled to the substrate to form thedipsticks.

C. Design of Dipsticks

A dipstick may contain more than one antibody so that the singledipstick can be used to detect more than one apolipoprotein orlipoprotein. For example, two or more separate pieces of a solid phasematerial, each coated with an antibody directed against a particularapolipoprotein or lipoprotein, can be attached to a longer strip ofsolid support to produce a dipstick with two or more separate areas,each specific for a particular lipoprotein or apolipoprotein. The meansto attach the solid phase material to a solid support should not impairthe function of the molecules coated on the solid phase material andmust be secure enough to withstand soaking in whole blood, serum,plasma, and the other solutions described herein which are used to wash,stain, and preserve the dipsticks. A preferred method of attachingantibody-coated solid phase material to a longer strip of solid supportis to use a glue or cement such an acrylate adhesive (for example, SUPERGLUE™, Super Glue Corporation, Hollis, N.Y.; DURO™, Loctite Corporation,Cleveland, Ohio).

Dipsticks can be designed for quantification of one or more lipoproteinsor apolipoproteins in a blood sample. In one embodiment, dipsticksdesigned for quantification of a lipoprotein or apolipoprotein contain asingle antigen-binding area which is dipped into a blood sample, stainedfor bound lipid lipoproteins or apolipoprotein, and visually comparedwith a set of printed colored standards to determine the concentrationof the particular lipoprotein or apolipoprotein.

In addition, dipsticks can be designed for detecting a change in therelative level of particular lipoproteins or apolipoproteins in a bloodsample. Dipsticks can be designed for detecting a change in the relativelevel of specific lipoproteins or apolipoproteins which contain twoantigen-binding areas, each area coated with a different antibody. Afterprocessing the dipstick to detect the lipoprotein or apolipoproteinantigens bound by each antibody, the relative intensities of the colorsin the two areas of the dipstick are compared as an indication of therelative concentrations of the two antigens in the blood.

A determination of relative levels of specific lipoproteins orapolipoproteins can also be made by simultaneously using two separatedipsticks. However, a single dipstick with two antigen binding areas isgenerally easier to use, especially for the lay person, and anassessment of relative color intensities in two areas in close proximityon a single dipstick is relatively easy to make even for the untrainedobserver.

A simple comparison of relative color intensities in two areas may besufficient for an assessment of an increase or decrease in a lipoproteinor apolipoprotein ratio. However, in a preferred embodiment, eachdoublet of stained areas is also compared with printed colored standardscovering an appropriate range of ratios of color intensities.

In another embodiment, dipsticks are made that contain distinct areas orspots of known amounts of molecules whose levels are to be determined bythe dipstick. For example, known amounts of lipid, lipoproteins and/orapolipoproteins are placed on the dipsticks using methods such as thoseused for attaching antibodies to the solid phase material describedabove. Such known amounts of lipids, lipoproteins, and apolipoproteinspresent on dipsticks act as “internal standards”, whose stainingintensity can be compared to that in the antigen-binding areas of thedipstick in order to estimate the amount of antigen bound by theantibodies on the dipstick.

D. Storage of Dipsticks

Although there is a possibility that some antibodies could be adsorbedonto a solid phase material, dried, and subsequently rehydrated withoutsignificant loss of their binding capacity, most of the antibodies ondipsticks are better preserved if stored in at least a small amount ofaqueous buffer, such as phosphate buffered saline (pH 7.4), in order toretain their binding capabilities. For example, the dipsticks can bestored damp in sealed foil or plastic bags containing enough buffer toprevent dehydration. The buffer may also contain an appropriate quantity(25 to 50 percent) of a stabilizing agent such as glycerol or sucrose.The dipsticks in the sealed bags can be stored in such buffers attemperatures ranging generally from 4 to 25° C., for up to three monthswithout significant loss of accuracy. The dipsticks should be removedfrom the storage bag immediately prior to use, and rinsed for 30 secondsunder tap water or physiological buffer (for example, PBS) (at atemperature less than 40° C. to avoid denaturation of the immobilizedMAb) in order to remove residual stabilizing agents and storage buffer.

III. Determination of Lipoprotein Concentrations

The crucial reagents in this approach are the antibodies or functionalfragments of the antibodies, which specifically recognize and bind aparticular lipoprotein, leaving other lipoproteins in the sampleunadsorbed. In order to assay a sample of whole blood, serum or plasmafor HDL or LDL, dipsticks are incubated with EDTA-treated or heparinizedblood for 2 to 5 minutes at room temperature. After incubation, eachstrip is washed to remove unbound blood, (for example, under tap waterfor 0.5 to 1 minute at temperatures not exceeding 40° C. The dipsticksare then stained, for example, by immersing the dipsticks in a solutionof stain such as Sudan Red 7B for 2 to 5 minutes at room temperature tostain the lipid present in the bound lipoprotein particles. Excess stainis then removed by an additional wash. Residual moisture or stain may bedrawn off by touching an absorbent towel with the edge of dipstick. The“face” of the dipstick, that is, the side of the dipstick containingimmobilized antibody, should not be blotted, which might disturb theimmobilized antibody and/or bound antigen. After drying, the intensityof the staining can be compared with standardized colored strips todetermine the concentration of lipoprotein in the blood.

A number of other lipid stains such as Oil Red O or Sudan Black B can bealso used for staining of dipsticks. However, in the preferredembodiment, Sudan Red 7B, also known as Fat Red 7B (Sigma, St. Louis,Mo.), dissolved in a mixture of methanol and NaOH is used because of itshigh color intensity. In another embodiment lipoproteins are stainedprior to being bound to antibody (“pre-stained”), such as antibody on adipstick, using any of the above mentioned lipid stains dissolved inpropylene glycol (Wollenweber, J. and Kahlke, W., Clin. Chim. Acta,29:411–420 (1970)). The pre-stained blood, plasma or serum sample isthen incubated, for example, with anti-LDL or anti-HDL dipsticks. Afterwashing and drying, the quantity of pre-stained lipoprotein captured bythe dipstick is determined visually according to the intensity of thecolor, for example, by comparison with a set of printed coloredstandards.

IV. Determination of Apolipoprotein Concentrations

Sandwich Assays

The methods described above for the detection of lipoproteins depend onthe staining of lipids associated with the lipoproteins which have beenbound by a lipoprotein-specific antibody on the dipstick. Thedetermination of the concentration of a specific apolipoprotein in bloodsamples requires a “sandwich” method of detection in which at least twoanti-apolipoprotein antibodies with distinct specificities for twodifferent epitopes of the same apolipoprotein are used. In a preferredembodiment, two MAbs are used that bind to separate epitopes of theapolipoprotein. One of the two MAbs is conjugated to an enzyme, forexample, horseradish peroxidase, alkaline phosphatase, or to biotinwhich in turn binds to an avidin- or streptavidin-enzyme conjugate of anenzyme-based chromogenic labeling system. The enzyme-conjugatedanti-apolipoprotein MAb is added to and mixed with the blood, serum orplasma sample. The second MAb is immobilized on a dipstick. During theincubation with the blood sample, typically 10 minutes at roomtemperature), the enzyme-conjugated MAb binds to its cognate epitope andforms soluble antibody-antigen complex. The dipstick is then immersedinto the blood sample and incubated for 2 to 5 minutes at roomtemperature to allow the immobilized MAb to bind the other epitope ofthe same apolipoprotein. The dipstick is then removed and washed asdescribed above. After washing, the strip is immersed into a solutioncontaining the appropriate chromogenic substrate (2 to 5 minutes at roomtemperature) for the enzyme that was conjugated to the first MAb forexample, 3,3′,5,5′-tetramethylbenzidine (“TMB”) or 4-chloro-1-naphtholfor horseradish peroxidase; or 5-bromo-4-chloro-3-indolyl phosphate foralkaline phosphatase. The dipstick is washed, dried, and the colordeveloped and compared with color standards which correspond to variousconcentrations of apolipoprotein in the blood sample.

Alternatively, the dipstick can first be incubated with a blood samplefor 2 to 5 minutes to bind the apolipoprotein and then washed andimmersed into the solution of the MAb-enzyme complex for 10 minutes atroom temperature. After an additional washing, the dipstick is immersedinto a solution of chromogenic substrate and stained as explained above.

For example, in a noncompetitive “sandwich” version of ELISA, anti-LDLMAb is adsorbed to the wells of a microtiter plate. A plasma or serumsample is added to wells of a microtiter plate coated with anti-LDLMAbs. The sample is incubated in the wells to allow the anti-LDL MAb tobind LDL in the plasma or serum sample. The unbound components of thesample are then removed and the quantity of LDL-Apo B captured by theHB₃cB₃ MAb is determined using a Pan B (D₆) MAb-peroxidase complex andchromogenic peroxidase substrate as described above. Peroxidase labeledpolyclonal antibody to Apo B may be used instead of Pan B MAb.

In a competitive variation of ELISA, anti-LDL HB₃cB₃ MAb is mixed with asample of plasma or serum and allowed to bind to LDL. This mixture isthen added to the ELISA microtiter plate coated with LDL. MAb moleculesthat did not react with LDL in the sample are free to bind to the layerof LDL immobilized on the plate. The higher the concentration of LDL isin the plasma or serum sample, the less anti-LDL MAb will bind to LDL onthe plate. The quantity of anti-LDL MAb bound to the plate is determinedusing any of the commercially available enzyme-conjugated secondaryantibodies such as, alkaline phosphatase or peroxidase conjugated togoat anti-mouse IgG, (Kirkegaard and Perry Laboratories, Gaithersburg,Md.), and subsequent incubation with an appropriate chromogenicsubstrate. Alternatively, the anti-LDL MAb can be conjugated to anenzyme, for example to form an anti-LDL MAb-peroxidase or alkalinephosphatase complex, and thereby eliminate the use of anenzyme-conjugated secondary antibody, with the same results.

In an alternate embodiment of the sandwich method, only one of theantibodies to the particular apolipoprotein is an MAb and the otherantibody is a polyclonal anti-apolipoprotein antibody. This method canwork as well as the two MAb sandwich method described above, if the oneMAb is specific for the particular apolipoprotein of interest, that is,does not cross-react with other apolipoproteins. Either the MAb or thepolyclonal antibody may be the immobilized or the enzyme-conjugatedantibody in this embodiment of the sandwich method. This method is mostaccurate when the MAb (whether enzyme-conjugated or immobilized) isallowed to bind to the apolipoprotein antigen first, and the polyclonalanti-apolipoprotein is allowed to bind to the apolipoprotein second.This stepwise procedure prevents underestimation of the quantity of theparticular apolipoprotein in the blood sample by insuring 1) that noneof the polyclonal antibody molecules are given the first opportunity tobind, and thereby block, the specific epitope recognized by the MAbmolecules and 2) that essentially only those apolipoprotein moleculesrecognized by the MAb are detected. The highly specific anti-Apo B MAbHB₃cB₃ described herein is thus an example of an MAb useful in any ofthe above-described sandwich methods as applied to the detection andquantification of Apo B.

The above described sandwich method, to determine the amount ofapolipoprotein in a sample, is useful not only for quantification ofsingle apolipoproteins, but also for analysis of ratios between variousapolipoproteins and lipoproteins in which case dipsticks with two ormore antigen binding areas are used as described above.

In another embodiment, any of the above described enzyme-conjugatedmonoclonal or polyclonal anti-apolipoprotein antibodies is replaced witha “stained” antibody, that is, an antibody coupled with a protein stainsuch as nitro blue tetrazolium (Glenner, G. G. Formazans and TetrazoliumSalts, In: H. J. Conn's Biological Stains, pp. 225–235. The Williams andWilkins Company USA 1990; U.S. Pat. No. 4,786,589)). Such a “stained” or“colored” antibody is mixed with a blood, plasma or serum sample tobind, and thereby, pre-stain the antibody's cognate apoliprotein orlipoprotein in the sample. An antibody coated dipstick is then immersedinto the sample in order to absorb the lipoprotein which has beenpre-stained via binding to the stained antibody. After washing anddrying the quantity of pre-stained lipoprotein is determined visually bycomparing the color of the dipstick with a set of printed colorstandards.

In addition, the above-described sandwich method can be used to detectany blood protein of interest in a particular sample, provided, asdescribed above, that either two distinct MAbs are available which donot interfere with each other's binding to the particular protein, orone MAb and a polyclonal antibody are available for the particularprotein and the MAb is allowed to bind to the particular protein beforethe polyclonal antibody.

Antibody-antigen Precipitation Techniques and Enzyme-linkedImmunosorbent Assays (ELISA)

Anti-LDL MAbs, such as HB₃cB₃ are useful for quantification ofLDL-cholesterol in antibody-antigen precipitation techniques andenzyme-linked immunosorbent assays (ELISA). For example, in aprecipitation method the anti-LDL MAb is added to human serum or plasmaand allowed to bind to LDL. The immune complex of LDL bound to anti-LDLMAb is then precipitated by mixing in an excess amount of protein A oran anti-mouse IgG polyclonal antibody. Precipitation of the complexes isenhanced by centrifuging the mixture and the supernatant liquid isdiscarded. The precipitate containing LDL is then washed and dissolvedin 8 M urea in PBS or treated with detergents such as Triton X-100 andcholic acid (Sigma, St. Louis, Mo.). This is followed by determinationof LDL-cholesterol using an enzymatic assay for cholesterol (Sigma, St.Louis, Mo.).

Fluorescent Immunoassay

Antibodies specific for LDL can be also used in fluorescent immunoassay.A number of fluorescent compounds such as fluorescein isothiocyanate,europium, lucifer yellow, rhodamine B isothiocyanate (Wood, P. In:Principles and Practice of Immunoassay, Stockton Press, New York, pages365–392 (1991), incorporated herein by reference) can be used to labelanti-LDL MAb or LDL. In conjunction with the known techniques forseparation of antibody-antigen complexes, these fluorophores can be usedto quantify LDL. The same applies to chemiluminescent immunoassay inwhich case either anti-LDL MAb or LDL can be labeled with isoluminol oracridinium esters (Krodel, E. et al., In: Bioluminescence andChemiluminescence: Current Status. John Wiley and Sons Inc. New York, pp107–110 (1991); Weeks, I. et al., Clin. Chem. 29:1480–1483 (1983),incorporated herein by reference). Radioimmunoassay (Kashyap, M. L. etal., J. Clin. Invest. 60:171–180 (1977)) is another technique in whichanti-LDL MAb can be used after coupling of anti-LDL MAb or LDL with aradioactive isotope such as ¹²⁵I. Some of these immunoassays can beeasily automated by the use of appropriate instruments such as the IMX™(Abbott, Irving, Tex.) for a fluorescent immunoassay and Ciba CorningACS 180™ (Ciba Corning, Medfield, Mass.) for a chemiluminescentimmunoassay.

V. Purification of Apolipoprotein

Although described herein with reference to the use of the specificanti-apolipoprotein antibodies for diagnostic purposes, the antibodiescan be immobilized to resins or well plates or other inert substratesfor use in purification of the apolipoprotein which the antibody isimmunoreactive with. Antibodies specific for LDL can also be used tomake immunoaffinity columns in which anti-LDL MAb is conjugated to asolid support. In addition to use in LDL purification, suchimmunoaffinity columns can be used to selectively remove LDL from apatient's blood using an extracorporeal circulation device. In apreferred embodiment, the antibody is bound to an acrylamide or agaroseresin particulate such as SEPHAROSE™ (Pharmacia Fine Chemicals,Piscataway, N.J.) or Affi-GEL™, (Bio-Rad, Hercules, Calif.), and placedin a chromatography column. The sample from which the apolipoprotein isto be purified is applied to the column, the material which is not boundby the antibody is washed from the column, and the bound lipoprotein iseluted from the antibody using a salt gradient or other standardtechnique.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Determination of Binding Specificity of Apolipoprotein MAbs

To determine the binding specificity of the anti-Apo A-I MAb AIbD5,ELISA plates were coated with antigens using concentrations indicated inthe left column of Table 1, below. Each antigen was incubated with theAIbD₅ MAb (10 μg/ml) followed by washing and detection of the bound MAbwith a goat anti-mouse-peroxidase conjugate. Each number represents anaverage optical density from three separate experiments. AIbD₅ MAb boundstrongly to Apo A-I and HDL, but exhibited no significant binding toother antigens.

TABLE 1 Binding Specificity of AIbD5 MAb (anti-Apo A-I) toapolipoprotein and lipoproteins. Antigen (μg protein ApolipoproteinsChylo- Lipoproteins per ml) A-I A-II C-III E microns VLDL LDL HDL 801.620 0.105 0.084 0.090 0.153 0.205 0.211 1.732 40 1.012 0.090 0.0600.071 0.095 0.132 0.130 1.361 20 0.841 0.080 0.053 0.060 0.080 0.0950.098 1.045 10 0.520 0.075 0.032 0.045 0.080 0.090 0.087 0.783 5 0.2100.063 0.040 0.038 0.075 0.080 0.075 0.400 2.5 0.135 0.020 0.020 0.0180.060 0.047 0.050 0.268

To determine the binding specificity of the Apo A-I MAb, AIbE₂, ELISAplates coated with antigen concentrations indicated in the left columnof Table 2, below. As in the case of AIbD₅ MAb, each antigen wasincubated with the AIbE₂ MAb (10 μg/ml) followed by washing anddetection of the bound MAb with a goat anti-mouse-peroxidase-conjugate.The numbers represent average optical density readings from threeseparate experiments. AIbE₂ bound strongly to Apo A-I and HDL, butexhibited no significant binding to the other antigens.

The epitopes recognized by the MAbs AIbD₅ and AIbE₂ are different andsterically separated since these antibodies did not compete with eachother when allowed to bind simultaneously to HDL.

TABLE 2 Binding to AIbE₂ MAb (anti-Apo A-I) to apolipoproteins andlipoproteins Antigen (μg protein Apolipoproteins Chylo- Lipoproteins perml) A-I A-II C-III E microns VLDL LDL HDL 80 1.205 0.095 0.061 0.0760.107 0.183 0.200 1.431 40 0.780 0.080 0.060 0.053 0.103 0.115 0.1631.108 20 0.337 0.083 0.065 0.060 0.098 0.108 0.108 0.860 10 0.340 0.0710.047 0.059 0.080 0.083 0.099 0.495 5 0.189 0.070 0.053 0.045 0.0630.070 0.063 0.231 2.5 0.105 0.068 0.048 0.040 0.060 0.058 0.060 0.150

EXAMPLE 2 Production of Anti-Apo B-100 Antibody, HB_(3c)B_(3.)

The MAb to Apo B, HB₃cB₃, was produced by immunizing mice with Apo B-100molecules which had been delipidized, reduced, carboxymethylated, andpurified by electrophoresis on a polyacrylamide gel containing 8 M urea.Delipidized Apo B-100 readily precipitates due to self-aggregation inaqueous media. In addition to the self-aggregation, Apo B-100 is alsosusceptible to fragmentation during the solubilization procedure(Socorro, L. and Camejo, G. J. Lipid Res., 20:631–645 (1979); Olofsson,S. O. et al., Biochemistry, 19:1059–1064 (1980)). Therefore, in order toseparate self-aggregated and degraded material from the preservedprotein, the delipidized, reduced, and carboxymethylated Apo B-100 waselectrophoresed on a polyacrylamide gel containing 8 M urea. Coomassieblue staining of the urea-polyacrylamide gel revealed three distinctbands. The most prominently stained band in the urea-containingpolyacrylamide gel was cut out immediately after the completion ofelectrophoresis and subcutaneously injected (while still in the gel)into mice without further manipulation of addition or adjuvants. Themost prominently stained band on the urea-polyacrylamide gel hadpreviously been shown to be pure Apo B-100, as confirmed by eluting theband from the urea-containing gel and electrophoresing it under reducingand denaturing conditions on a standard SDS-containing polyacrylamidegel. The SDS-gel revealed a single protein band of the expected mobilityof Apo B-100.

Approximately 10 to 20 μg of the Apo B-100 band excised from theurea-containing gel was injected four times at various locations over aperiod of two months. The mice immunized with the Apo B-100 according tothis procedure were then used in standard methods to produce hybridomas.Out of forty-two hybridomas which produced MAbs that bound Apo-B-100,only one, HB₃cB₃, produced a MAb that bound exclusively to LDL, as shownin Table 3, below.

To characterize the binding specificity of the HB₃cB₃ MAb, ELISA plateswere coated with lipoproteins using concentrations indicated in the leftcolumn of Table 3 below. Each antigen was incubated with the HB₃cB₃ MAb(10 μg/ml) followed by washing and detection of the bound MAb with agoat anti-mouse IgG-peroxidase conjugate. Each number represents anaverage optical density reading from three separate experiments. HB₃cB₃MAb showed a strong and exclusive binding to LDL. Identical results wereobtained with competitive ELISA (see below) in which the binding ofHB₃cB₃ MAb to LDL absorbed to the wells of an ELISA plate was found tobe inhibited only by LDL.

TABLE 3 Binding Specificity of HB₃cB₃ MAb (anti-Apo B-100) toLipoproteins Antigen Lipoproteins (μg protein Chylo- per ml) micronsVLDL LDL HDL 80 0.085 0.098 1.900 0.078 40 0.081 0.095 1.432 0.080 200.072 0.084 1.003 0.082 10 0.060 0.068 0.605 0.075  5 0.043 0.063 0.2110.060  2.5 0.040 0.051 0.140 0.060

HB₃cB₃ MAb binds to Apo B-100 in Western blots and shows no significantreactivity with any other plasma apolipoproteins or proteins. Westernblotting also reveals that HB₃cB₃ MAb binds to the so-called T₂ fragmentof Apo B-100 which represents a carboxy terminal 1,287 amino acid pieceof Apo B-100 (Cardin, A. D. et al. J. Biol. Chem., 259: 8522–8528(1984)). The HB₃cB₃ MAb recognizes an epitope outside of the receptorbinding domain localized at the amino terminus of the T₂ fragmentbecause it does not interfere with the binding of LDL to the LDLreceptor on cultured human skin fibroblasts and human hepatoma HepG2cells.

HB₃cB₃ MAb binds strongly and specifically to LDL with little or nosignificant reactivity with VLDL (Table 3, above). Furthermore,immunoaffinity chromatography of human serum using HB₃cB₃ MAbimmobilized on an AFFI-GEL™ column (Bio-Rad, Hercules, Calif.) alwaysyields a lipoprotein fraction with typical β electrophoretic mobility,free of any other lipoproteins. Identical results were obtained withnormal, hypertriglyceridemic as well as hypercholesterolemic sera asdetermined using a commercial lipoprotein electrophoresis kit (CibaCorning, Medfield, Mass.). In addition, crossed immunoelectrophoresis(Koren, E., et al., Biochemistry, 21:5347–5351 (1982)), of thelipoproteins retained by the HB₃cB₃ column revealed only one symmetricalApo B peak very similar in shape and mobility to ultracentrifugallyisolated LDL. These immunoaffinity chromatography results confirmed thespecificity of HB₃cB₃ MAb for LDL as well as the lack of reactivity withVLDL.

Further evidence for the LDL specificity of HB₃cB₃ MAb came from acomparison between the LDL-Apo B concentrations as determined in humansera using an ELISA with HB₃cB₃ MAb and the concentrations ofLDL-cholesterol determined using a commercially availableLDL-cholesterol assay kit (Sigma, St. Louis, Mo.). In the competitiveELISA method, the wells of microtiter plates were coated with LDL andblocked with 0.1% nonfat milk proteins (Kirkegaard Perry Laboratories,Gaithersburg, Md.). This was followed by incubating dilutions of humansera with HB₃cB₃ MAb for 18 hours at 4° C. These mixtures were thenpipetted into wells coated with LDL and incubated for 3 hours at roomtemperature. During this time, HB₃cB₃ MAb molecules that did notpreviously bind to LDL in the serum, bound to the LDL coating the plate.The quantity of HB₃cB₃ MAb that was bound to LDL on the plate wasinversely proportional to the concentration of LDL in the serum sample.After washing off the unbound components, bound HB₃cB₃ was detectedusing peroxidase-labeled anti-mouse IgG and the chromogenic (ABTS)peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg,Md.). Intensity of developed color was determined using an ELISA platereader MR 580 (Dynatech, Chantilly, Va.). Dilutions of pure LDL(isolated by ultracentrifugation, see Alaupovic, P. et al., Biochim.Biophys. Acta, 260: 689–707 (1972), incorporated herein by reference)with known concentrations of ApoB-100 were used on each plate toconstruct a standard curve from which the concentrations of LDL-Apo B inthe serum samples were calculated. The LDL-cholesterol concentrationswere determined in the same sera using the LDL-DIRECT™ commercial kit(Sigma, St. Louis, Mo.). This method was used because it allows for anaccurate determination of LDL-C even in sera with triglycerides as highas 1139 mg/dl.

Based on analysis of 100 human sera with variable lipoprotein profiles,the correlation between LDL-Apo B values determined by the HB₃cB₃ ELISAand LDL-cholesterol values determined by the commercial kit was highlysignificant. The correlation coefficient was 0.94 and corresponding Pvalue was <0.0001. Twenty-six of these sera contained very high levelsof triglycerides (400 to 1125 mg/dl), and were therefore rich in VLDL.However, the presence of excess VLDL did not interfere with theselective recognition of LDL by the HB₃cB₃ MAb. The correlation betweenLDL-Apo B determined by the HB₃cB₃ ELISA and the LDL-C determined by thecommercial kit (Sigma, St. Louis, Mo.) was highly significant (r=0.93,p<0.0001)) even in this subgroup of twenty-six hyper-triglyceridemicsera.

The above data clearly indicated that HB₃cB₃ MAb recognizes an epitopeof Apo B-100 that is fully expressed only on LDL particles. The HB₃cB₃hybridoma cells producing the antibody were deposited in the AmericanType Culture Collection (12301 Parklawn Drive, Rockville, Md. 20852)under the ATCC designation number HB1161.

EXAMPLE 3 Preparation of Anti-LDL and Anti-HDL Dipsticks to Assay HumanSerum, Plasma and Whole Blood

To prepare dipsticks for analyses of LDL and HDL in human whole blood,serum and plasma samples, PVDF membrane (Bio-Rad, Hercules, Calif.) wastreated with methanol and washed with water according to manufacturer'sinstructions. Washed membrane was cut into strips (5×60 mm) and storedin phosphate buffered saline (Sigma, St. Louis, Mo.) pH 7.4 at 4° C. Thestrips were incubated with the anti-LDL MAb HB₃cB₃ or the anti-HDL MAbAIbD₅ in PBS. Both of these MAbs were adjusted to the concentration of 1mg/ml. Each strip was incubated in 6 ml of an antibody solution for 24hours at 4° C. followed by two additional 24-hour incubations usingfresh antibody solutions each time to adsorb MAbs to each strip. Thepurpose of these sequential incubations was to saturate strips withadsorbed antibodies. This was accomplished by the three consecutiveincubations as indicated by the concentrations of antibodies left insolution after each incubation with PVDF strips. Coating of strips withMAbs was followed by an incubation in a 2% solution of nonfat milkproteins in PBS (Kirkegaard and Perry Laboratories, Gaithersburg, Md.)for 24 hours at 4° C. to block areas of PVDF not occupied by antibodymolecules. After three washes in 30 ml of PBS, the strips were kept inPBS at 4° C. up to two weeks without noticeable loss of activity.Antibody-coated and blocked strips were also immersed in PBS containing50 percent sucrose (Sigma, St. Louis, Mo.) for 5 minutes and sealed insmall plastic bags. The sealed dipsticks retained their capacity to bindlipoproteins for up to twelve weeks at 4° C.

EXAMPLE 4 Use of Dipsticks to Assay LDL and HDL by Lipid staining andComparison to Other Assay Method

Samples of human blood serum or plasma were diluted with 0.5% EDTAsolution by adding 100 μl of EDTA to 100 μl of sample in an 0.5 mlplastic tube. A small piece of antibody-coated dipstick (5×5 mm) wasimmersed into diluted serum for 2 minutes at room temperature. The tubewas shaken occasionally two to three times. This was followed by washingoff unbound constituents of serum with tap water for 1 minute. The stripwas then gently blotted against paper tissue to remove excess water andair dried for 2 minutes. Washing and drying was followed by staining ofthe strip in 200 μl of 0.02% Sudan Red 7B (Sigma, St. Louis, Mo.)dissolved in a mixture of methanol and 0.1 M NaOH (5:1 volume: volume)for 3 minutes with occasional shaking. The staining was followed bywashing under the tap water for 1 minute, blotting and air drying for 5minutes. A dipstick coated with no antibody and blocked with nonfat milkproteins served as a negative control. The whole procedure lastedapproximately 15 minutes and was carried out at room temperature. SudanRed 7B stained the lipid moiety of lipoproteins (LDL and HDL,respectively) captured on the antibody-coated strips. The intensity ofcolor was clearly proportional to concentrations of LDL and HDLcholesterol determined in each serum by the respective conventionalmethods (Sigma, St. Louis, Mo.), as shown in Tables 4 and 5 below.

In Table 4, serum LDL-cholesterol was determined in all samples by adirect LDL-C assay (Sigma, St. Louis, Mo.). The same serum samples alsowere incubated with anti-LDL dipsticks and stained with Sudan Red 7B asdescribed above. The color intensity was assessed visually (on anarbitrary scale of 1 to 15) by three individuals (I, II and III)presented with the complete set of 16 dipsticks at the same time. Theaveraged score of the color intensity correlated significantly withLDL-C concentrations (r=0.97, p<0.0001).

TABLE 4 Correlation between the serum LDL-cholesterol concentration andthe color intensity of HB₃cB₃ coated (anti-LDL) dipsticks stained withSudan Red 7B. Serum LDL- Sample cholesterol Dipstick color intensityAverage number (mg/dl) I II III color score 1 145 4 4 4 4.0 2 130 4 4 33.7 3 97 2 2 2 2.0 4 165 6 5 4 5.0 5 115 3 2 2 2.3 6 200 8 10 9 9.0 7207 8 8 9 8.3 8 160 6 5 5 5.3 9 115 2 3 2 3.3 10 276 15 15 15 15.0 11155 6 5 5 5.3 12 98 2 1 1 1.3 13 123 3 3 3 3.0 14 130 3 4 2 3.0 15 185 66 7 6.3 16 73 1 1 1 1.0

In Table 5, serum HDL-cholesterol was determined in all samples bySigma's HDL-C kit. The same serum samples were incubated with anti-HDLdipsticks and stained with Sudan Red 7B as described above. The colorintensity was assessed visually (on an arbitrary scale of 1 to 10) bythree individuals (I, II and III) presented with the complete set of 9dipsticks at the same time. The averaged score of the color intensitycorrelated significantly with HDL-C concentrations (r=0.93, p<0.0005)

TABLE 5 Correlation between the serum HDL-cholesterol concentration andthe color intensity of AIbD₅ coated (anti-HDL) dipsticks stained withSudan Red 7B. Serum LDL- Sample cholesterol Dipstick color intensityAverage number (mg/dl) I II III color score 1 65 10 10 10 10.0 2 53 6 66 6.0 3 58 5 6 6 5.7 4 48 5 5 6 5.3 5 40 5 6 5 5.3 6 60 7 8 8 7.7 7 28 11 1 1.0 8 37 3 3 3 3.0 9 39 3 3 3 3.0

Two other lipid stains, Oil Red O and Sudan Black B, gave similarresults. All of these stains are commonly used for staining oflipoproteins in electrophoretic analyses of serum (Stein E. A. andMeyers, G. L., Lipids, Lipoproteins and Apolipoproteins, In TietzTextbook of Clinical Chemistry, W. B. Saunders, Philadelphia pp1002–1093 (1994)). The color on dipsticks is stable for fourteen days.Anti-LDL dipsticks were more intensely colored than anti-HDL dipstickswhich reflects higher lipid content per LDL particle. The aboveexperiments were also carried out with human plasma and serum withidentical results. The total time to run the dipstick assay, frominsertion into a blood sample to development of color is approximately15 minutes.

EXAMPLE 5 Stability of Dipsticks and Lipid Stains

Anti-LDL and anti-HDL dipsticks were stored in 50 percent sucrose insealed plastic bags for 3, 6 and 12 weeks at 4° C. as described above.Dipsticks were washed under the tap water for 1 minute and usedimmediately after removal from plastic bags. Incubations with serum andstaining were carried out as described above at each of the indicatedtime intervals. To avoid storage-related decline in serum lipoproteinsconcentrations, aliquots of serum were stored at −70° C. and thawed atindicated time intervals immediately prior to experiments withdipsticks. There were no noticeable differences between anti-LDLdipsticks stored for various times over a period of twelve weeks.Similar results were obtained with anti-HDL dipsticks. All three lipidstains (Sudan Red 7B, Sudan Black B and Oil Red O) dissolved in methanolwere stable for four months at room temperature as well as 4° C.However, the 0.1 M NaOH solution, which is present in each stainingsolution, must be added immediately prior to staining the dipsticks toassure optimal and reproducible results.

EXAMPLE 6 Use of the Avidin-Biotin Complex to Bind Antibody to Dipsticks

The avidin-biotin system was also used to bind antibody molecules toPVDF strips. PVDF strips were incubated with egg-yolk avidin (Sigma, St.Louis, Mo.) dissolved in PBS (3 mg/ml) for 24 hours at 4° C. This stepwas repeated for a total of three times using fresh avidin solution eachtime. Strips were then blocked with 2% nonfat milk for 24 hours at 4° C.The strips were then incubated three times for 24 hours at 4° C. in asolution of biotinylated anti-LDL HB₃cB₃ MAb (1 mg/ml). The anti-LDL MAbwas biotinylated at its Fc fragment using theperiodate-biotin-LC-hydrazide technique (Pierce, Rockford, Ill.) whichcovalently couples biotin molecules to carbohydrate residuesconcentrated at the Fc portion of the antibody molecule. Biotinylationcarried out in this fashion leaves the antigen combining sites of anantibody intact. Furthermore, with the Fc portion of the antibodyattached to the layer of avidin on the strip, the antigen-binding sitesare free to bind the antigen.

The avidin-biotin anti-LDL strips made with the avidin-biotin complexwere kept in PBS at 4° C. These anti-LDL dipsticks made withavidin-biotin complexes stained more intensely than dipsticks coatedwith non-biotinylated anti-LDL. Each avidin molecule consisting of foursubunits can bind four biotin molecules and the affinity of bindingbetween these two molecules is extremely high (Savage, M. D. et al.Avidin-Biotin Chemistry: A Handbook, Pierce Chemical Company, Rockford,Ill. (1992)). Thus, anti-LDL dipsticks made with avidin-biotin complexesexhibited a higher LDL binding capacity than dipsticks made without theavidin-biotin system to bind antibody to the dipsticks.

EXAMPLE 7 Dipstick Method to Assay LDL by Staining of Apolipoprotein B

The Pan B D₆ MAb is specific for Apo B and binds equally well to all ApoB-containing lipoproteins including LDL. The binding of Pan B (D₆) MAbto LDL does not interfere with the binding of HB₃cB₃ MAb to LDL. D₆binds to the amino terminal half of Apo B-100 whereas HB₃cB₃ binds tothe carboxy terminal end of B-100. Thus, both MAbs can bindsimultaneously to the same LDL particle due to sufficient stericdistance between their corresponding epitopes. The Pan B (D₆) Mab,biotinylated at the Fc fragment as described above, was mixed withstreptavidin-peroxidase (BRL, Bethesda, Md.) to form antibody-peroxidasecomplex due to binding between the biotin on the antibody and thestreptavidin conjugated to peroxidase. The complex was dialyzed againstPBS containing 25% sucrose (Sigma, St. Louis, Mo.). Thisperoxidase-tagged Pan B (D₆) MAb complex was still capable ofrecognizing and binding to LDL. PVDF strips were coated with anti-LDLantibody (HB₃cB₃) and blocked as described above. Anti-LDL dipstickswere incubated with human serum samples for 2 minutes, washed under tapwater, air dried for 2 minutes and incubated with biotinylated Pan B(D₆) MAb and streptavidin-peroxidase for 10 minutes. After theincubation with the Pan B (D₆) MAb-peroxidase complex, the dipstickswere washed with tap water and incubated for 2 minutes with thechromogenic peroxidase substrate TMB (Kirkegaard and Perry Laboratories,Gaithersburg, Md.). This was followed by an additional 1-minute washingunder tap water and drying at room temperature for 5 minutes. The Pan(D₆) MAb-peroxidase complex bound to LDL captured by the anti-LDL stripand converted TMB substrate into a colored compound. The whole procedurewas carried out at room temperature. The intensity of the blue-greencolor of dipsticks was proportional to the concentrations ofLDL-cholesterol in the respective serum samples as shown by the data inTable 6 below.

In Table 6, serum LDL-C was determined in all samples by direct LDL-Cassay (Sigma, St. Louis, Mo.). The same serum samples were incubatedwith anti-LDL dipsticks followed by incubation with D₆ MAb-peroxidaseand staining with TMB substrate as described above. The color intensitywas assessed visually (on an arbitrary scale of 1 to 15) by threeindividuals (I, II and III) presented with the complete set of 16dipsticks at the same time. The averaged score of the color intensitycorrelated significantly with LDL-C concentrations (r=0.98, p<0.0001).

TABLE 6 Correlation between the serum LDL-cholesterol concentration andthe color intensity of HB₃cB₃ coated (anti-LDL) dipsticks stained withD₆ MAb-peroxidase-TMB system. Serum LDL- Sample cholesterol Dipstickcolor intensity Average number (mg/dl) I II III color score 1 145 5 4 44.3 2 130 4 3 5 4.0 3 97 2 2 3 2.3 4 165 7 7 6 6.7 5 115 3 2 3 2.7 6 2008 9 9 8.7 7 207 8 9 9 8.7 8 160 5 6 7 5.7 9 115 2 2 2 2.0 10 276 15 1515 15.0 11 155 6 4 4 4.7 12 98 1 2 2 1.7 13 123 3 3 3 3.0 14 130 3 4 43.7 15 185 6 6 9 7.0 16 73 1 1 1 1.0

To summarize, the above experiments demonstrated that anti-LDL dipsticksallow for quantification of LDL by visualizing either lipids by stainingwith lipid stains or Apo-B by staining with a protein stain orchromogenic assay using a substrate such as TMB.

EXAMPLE 8 Dipstick Method to Assay HDL by Staining of Apolipoprotein A-I

Anti-HDL dipsticks coated with AIbD₅ MAb were used to adsorb HDLparticles in human serum samples as described above. After washing, thedipsticks were incubated with the second MAb to Apo-I (AIbE₂) complexedwith streptavidin peroxidase as described above. The AIbE₂-peroxidasecomplex bound to the HDL particles which were captured on the dipstickby the AIbD₅ MAb. After incubation with TMB, washing, and drying, thecolor intensity was proportional to the serum HDL cholesterol as shownby the data in Table 7 below.

In Table 7, serum HDL-cholesterol was determined in all samples by acommercial kit (Sigma, St. Louis, Mo.). The same serum samples wereincubated with anti-HDL dipsticks followed by incubation with AIbE₂MAb-peroxidase complex and staining with TMB as described above. Thecolor intensity was assessed visually (on an arbitrary scale of 1 to 10)by three individuals (I, II and III) presented with the complete set of9 dipsticks at the same time. The averaged score of the color intensitycorrelated significantly with HDL-C concentrations (r=0.97, p<0.0001)

TABLE 7 Correlation between the serum HDL-cholesterol concentration andthe color intensity of AIbD₅ (anti-HDL) dipsticks stained with AIbE₂MAb-peroxidase-TMB system Serum LDL- Sample cholesterol Dipstick colorintensity Average number (mg/dl) I II III color score 1 65 10 10 10 10.02 53 6 7 6 6.3 3 58 6 7 7 6.7 4 48 6 5 5 5.3 5 40 5 4 5 4.7 6 60 8 8 88.0 7 28 1 1 1 1.0 8 37 4 4 4 4.0 9 39 4 4 4 4.0

Both Pan B (D₆) MAb- and AIbE₂MAb-peroxidase complexes were stable inPBS containing 25% sucrose for at least 3 months at 4° C.

EXAMPLE 9 Dipstick Method to Assay the LDL/HDL Ratio

Anti-LDL and anti-HDL dipsticks, prepared as described above, were usedto determine the relative ratio of LDL-Apo B to HDL-Apo A-I, that is,the LDL/HDL ratio. Small pieces (0.5×0.5 cm) of both anti-HDL andanti-LDL dipsticks were simultaneously incubated with the same sample ofhuman serum, plasma, or whole blood for 2 minutes, washed under tapwater, air dried for 2 minutes and incubated with an equimolar mixtureof D₆ MAb- and AIbE₂ MAb-streptavidin-peroxidase complexes for 10minutes to detect bound LDL and HDL, respectively. This was followed bywashing under tap water, a 2-minute air drying, and a 2-minuteincubation with the TMB substrate as described above. After anadditional washing under tap water and air drying at room temperature (5minutes) the color intensity on both dipsticks was compared visually.The serum, plasma or blood samples with known concentrations ofHDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) were analyzed by theabove dipstick method. Sera with LDL-C concentrations between 110 mg/dland 130 mg/dl and HDL-C concentrations between 40 and 55 mg/dl showedcomparable color intensity on both dipsticks. Sera with LDL-C valueshigher than 140 mg/dl generally show more intense color on anti-LDLdipsticks. The only exceptions were the sera with HDL-C levels higherthan 50 mg/dl. In these cases, anti-HDL dipsticks tended to be moreintensely stained unless the LDL-C levels exceeded 160 mg/dl.

Virtually identical results were obtained when anti-HDL and anti-LDLdipsticks were used separately. In these experiments each serum withpreviously determined HDL-C and LDL-C was separately incubated withanti-HDL and anti-LDL dipsticks and stained with AIbE₂ and D₆MAb-peroxidase complexes, respectively. The agreement between these twotypes of experiments demonstrates that even in case of simultaneousincubation of anti-HDL and anti-LDL dipsticks with the same serum,plasma or blood sample followed by the simultaneous incubation of bothdipsticks with the mixture of AIbE₂ MAb- and D₆ MAB-peroxidase, thereactions between lipoproteins and corresponding MAbs coated on thedipsticks remain specific. HDL particles always bind to the AIbD₅ MAbcoating the anti-HDL dipstick and the AIbE₂ MAb-peroxidase complex bindsto the HDL-Apo A-I captured by the anti-HDL dipstick. The same is truefor LDL particles which bind exclusively to the HB₃cB₃ MAb coating theanti-LDL dipstick and react with the D₆ MAb-peroxidase complex.

These studies demonstrate that the dipstick methodology provides a quickand simultaneous determination of relative quantities of HDL and LDL inserum, plasma, or whole blood samples. To determine the LDL/HDL ratio inan unknown blood sample, the color intensities on the HDL and LDLdipsticks, which were incubated with the unknown sample, are compared toa set of printed color standards derived from blood samples with knownLDL/HDL ratios.

EXAMPLE 10 Dipstick Method to Assay LPA-I/LPA-I:A-II Ratio

To determine the LP A-I/LP A-I:A-II ratio, two dipsticks were used. Oneof them was coated with AIbD₅ MAb (anti-Apo A-I) and the other with CdB₅MAb (anti-Apo A-II). In addition to these two MAbs, a third MAb, AIbE₂(anti-Apo A-I) was also used. AIbE₂ was biotinylated at the Fc portionof IgG molecule and complexed with streptavidin-peroxidase as describedabove. Samples of EDTA treated whole blood, serum and plasma weresimultaneously incubated with both anti-Apo A-I and anti-Apo A-IIdipsticks for 2 minutes at room temperature. Dipsticks were then washedunder tap water, incubated with the AIbE₂ MAb-peroxidase complex for 10minutes, washed again, incubated with TMB substrate, washed and airdried as described above. Intensities of the blue-green color developedon both dipsticks were compared visually. The dipstick coated with AIbD₅MAb (anti-Apo A-I) captured both LP A-I and LP A-I:A-II particles, andit was always more intensely stained relative to the CdB₅ (anti-ApoA-II) coated dipstick. The latter dipstick captures only LP A-I:A-IIsubfraction which represents approximately 60% of all Apo A-I-containingparticles (Koren, E. et al. Clin. Chem., 33:38–43 (1987)). However,there were clear differences between various blood samples.

For example, in males, the difference between AIbD₅ (anti-Apo A-I)dipsticks and the CdB₅ (anti-Apo A-II) dipsticks, although present, weregenerally less noticeable due to somewhat weaker staining of theanti-Apo A-I dipsticks. In females, AIbD₅ dipsticks were usually moreintensely stained relative to CdB₅ coated dipsticks, reflecting higherconcentrations of Lp A-I in their blood (Koren, E. et al., Clin. Chem.,33:38–43 (1987)). In addition to these observations, there was a goodcorrelation between the relative color intensities of both dipsticks andtheir respective particles determined by the ELISA described by Koren,E. et al. Clin. Chem. 33:38–43 (1987), incorporated herein by reference.These experiments demonstrate that the dipstick methodology can besuccessfully used for a quick determination of LP A-I/LP A-I:A-II ratio.To determine the LP A-I/LP A-I:AII ratio in an unknown blood sample, thecolor intensities of the anti-Apo A-I and anti-Apo A-II dipsticks whichwere incubated with the unknown sample are compared to a set of printedcolor standards derived from dipsticks incubated with blood samples withknown concentrations of LP A-I and LP A-I:A-II.

EXAMPLE 11 Dipstick Method to Assay the Distribution of Apo C-III andApo-E (C-III Ratio and E Ratio)

The “C-III Ratio” has been shown to be a reliable indicator of theprogression of coronary artery disease (Alaupovic, P. and Blankenhorn,D. H. Klin. Wochenschr., 60:38–40 (1990); Blankenhorn, D. H. et al.Circulation, 81:470–478 (1990)). The current methodology for the C-IIIratio is based on precipitation of all Apo B-containing lipoproteinparticles with heparin and quantification of Apo C-III in both theheparin precipitate and heparin supernatant fraction. The Apo C-III inthe heparin precipitate fraction represents Apo C-III associated withApo B in VLDL and VLDL remnant particles. Apo C-III remaining in thesupernatant fraction is associated with HDL particles. The C-III ratiois calculated by dividing the Apo C-III in the heparin supernatant bythe Apo C-III in the heparin precipitate. A low C-III ratio isassociated with progression of coronary disease.

The dipstick methodology described above was also used to determine thedistribution of Apo C-III, that is, to obtain a C-III ratio. PVDF stripswere coated with the Pan B (D₆) MAb, blocked and incubated with humanserum plasma or whole blood as described above. This was followed bywashing, an incubation with the XbA₃ (anti-Apo C-III) MAb-peroxidasecomplex, an additional washing, and an incubation with chromogenic TMBsubstrate as described above. The color developed on the Pan B (D₆)MAb-coated dipsticks was proportional to the Apo C-III associated withApo B. As described earlier, the Pan B (D₆) MAb binds all ApoB-containing particles, including LDL. However, the amount of Apo C-IIIassociated with Apo B in LDL is negligible. Therefore, the colorintensity on the Pan B coated dipsticks reflected the amount of ApoC-III associated with VLDL and VLDL remnant particles. The anti-Apo A-I(or anti-HDL) dipsticks coated with AIbD₅MAb were also used incombination with the XbA₃ MAb-peroxidase complex. The color on thesedipsticks was proportional to the amount of Apo C-III associated withApo A-I in HDL particles.

A visual comparison of the Pan B and the anti-HDL dipsticks afterincubation with the same serum sample and staining, allowed for anestimation of the C-III ratio. A serum with a high C-III ratio (asdetermined by assaying C-III in a heparin supernatant and precipitate)showed relatively strong color on the anti-HDL dipstick and only a faintcolor on the Pan B (D₆) MAb dipstick. A serum, which was previouslyshown to have a low C-III ratio, showed more intense color on the Pan B(D₆) MAb dipstick and relatively weak color on the anti-HDL MAbdipstick. Identical results were obtained with whole blood.

Similar experiments were carried out to determine the Apo E ratio by theuse of appropriate dipsticks. As described above, the Apo E ratio isdetermined by dividing Apo E in heparin supernate with the Apo E inheparin precipitate. The Apo E ratio is analogous to the C-III ratio andreflects the quantity of VLDL and their remnants relative to the HDLparticles. To determine the Apo E ratio, the Pan B and anti-HDLdipsticks (coated with D₆ and AIbD₅ MAbs, respectively) were incubatedwith human serum, plasma or whole blood for 2 minutes, washed under tapwater, air dried for 2 minutes and incubated with an equimolar mixtureof two anti-Apo E MAbs (each complexed with streptavidin-peroxidase) for10 minutes. This was followed by washing and incubation with TMBsubstrate as described above. Two anti-Apo E MAbs (EfB₁ and EfD₃) wereseparately biotinylated at their Fc fragments and complexed with thestreptavidin-peroxidase as described above. Since EfB₁ MAb bindspredominantly to Apo E associated with VLDL, whereas EfD₃ preferentiallybinds Apo E on HDL particles, an equimolar mixture of EfB₁ andEfD₃-peroxidase complexes was used for incubation with the Pan B andanti-HDL dipsticks. This mixture was used in place of a single MAb withequal binding to all Apo E-containing lipoproteins. Nevertheless,determination of the Apo E ratio with the above dipsticks is quitesimilar to the Apo C-III ratio. The sera with low Apo E ratio determinedby the heparin precipitation method gave a relatively weak staining onanti-HDL dipsticks, reflecting a low concentration of Apo E associatedwith Apo A-I in HDL particles, and an intense staining on the Pan Bdipsticks due to the high concentration of Apo E associated with Apo B.The sera with high Apo E ratio gave an inverse pattern (strong stainingof anti-HDL and weaker staining of Pan B dipsticks).

These experiments demonstrate that a dipstick technique using describedcombinations of MAbs to apolipoproteins A-I, B, C-III and E provides aquick (approximately 30 minutes) estimation of the C-III and E ratios inhuman serum, plasma and whole blood, or other biological samples. Theconventional determinations of these ratios cannot be done with wholeblood and take 12–24 hours (Alaupovic, P. Can. J. Biochem., 59:565–579(1981)).

EXAMPLE 12 Production of Recombinant Anti-LDL Antibody

Murine hybridoma cells producing anti-LDL HB₃cB₃ MAb were used toproduce recombinant anti-LDL using a commercially available recombinantphage antibody system (RPAS, Pharmacia Biotech Inc., Piscataway, N.J.).Briefly, mRNA was isolated from HB₃cB₃ producing hybridoma cells,followed by synthesis of cDNA encoding the variable regions of bothheavy and light chains of the HB₃cB₃ MAb. The heavy and light chainencoding cDNAs were amplified in two separate PCRs using two sets ofprimers, specific for each chain. The amplified heavy and light chaincDNA fragments were then purified using agarose gel electrophoresis andassembled into a single recombinant DNA fragment using a DNA linkerfragment (Pharmacia Biotech Inc., Piscataway, N.J.). The resultingrecombinant DNA molecule encodes a single chain polypeptide, called asingle chain Fv fragment (ScFv), which binds the same epitope as theoriginal MAb.

The recombinant DNA fragment was approximately 700 base pairs in length.The assembled ScFv DNA was amplified with a set of oligonucleotideprimers that introduced the SfiI and NotI restriction sites. Thisrecombinant DNA fragment was further purified and sequentially digestedwith SfiI and NotI to generate cohesive ends for ligation into the phageplasmid (phagemid) pCANTAB 5 (Pharmacia Biotech Inc., Piscataway, N.J.)cloning vector. The inserted recombinant DNA encoding the ScFv was fusedwith the 5′ end of the gene coding the g3p minor coat protein located atthe tip of the phage. The ligated phagemid vector containing theinserted DNA, was introduced into competent E. coli TG 1 cells.

Phagemid-containing bacterial colonies were infected with M13 K07 helperphage to yield recombinant phage which display ScFv antibodies. At thisstage, recombinant anti-LDL ScFv antibody is expressed on the tip of thephage as a fusion product between the antigen-binding site of HB₃cB₃ MAband the M13 g3p minor coat protein. Phage, containing phage-displayedScFv antibodies capable of binding LDL, were selected by panning inLDL-coated cell culture flasks. The panning and reinfection of E. coliTG 1 cells was repeated several times until phage-displayed ScFvantibodies of high affinity were obtained. The LDL binding affinity ofthe ScFv antibodies was determined using an ELISA method. The wells ofmicrotiter ELISA plates were coated with serial dilutions (80 to 2.5μg/ml) of LDL and blocked as described above. Phage displaying anti-LDLScFv antibodies were pipetted into duplicate wells and allowed to bindfor 3 hours at room temperature (approximately 25° C.). After washing,the peroxidase-labelled sheep antibody directed against the M13 g8pmajor coat protein was added to detect the presence of recombinant phageantibodies bound to LDL. After washing, a peroxidase chromogenicsubstrate (ABTS, Kirkegaard and Perry Laboratories, Gaithersburg, Md.)was added and the resulting color intensity measured by the use of anELISA plate reader (MR 580, Dynatech Chantilly, Va.). The serialdilutions of LDL gave rise to a binding curve for each recombinant phageanti-LDL. The slopes of binding curves were compared to the slope of thenative anti-LDL HB₃cB₃ MAb which was used on each plate as a positivecontrol. Out of 35 phage ScFv antibodies, several showed affinitiescomparable to HB₃cB₃ MAb based on the slopes of binding curves as shownin Table 8.

In Table 8, ELISA plates were coated with LDL using concentrationsindicated in the left column. LDL coated wells were incubated (induplicates) with HB₃cB₃ MAb (2 μg/ml) and RcB₃M₁D₄ recombinant phageantibody as described above. Detection of bound antibodies was carriedout using the respective peroxidase labeled conjugates as describedabove. The numbers represent average optical density readings valuesfrom two separate experiments.

TABLE 8 Binding of HB₃cB₃ MAb (anti-LDL) and RcB₃M₁D₄ recombinant phageantibody to LDL. LDL concentration HB₃cB₃ monoclonal RcB₃M₁D₄recombinant μg protein/ml antibody Phage antibody 80 0.802 0.675 400.497 0.406 20 0.263 0.211 10 0.115 0.098 5 0.060 0.047 2.5 0.042 0.036

ScFv phage antibody with the highest affinity (RcB₃M₁D₄) was placed ondeposit at the American Type Culture Collection (12301 Parklawn Drive,Rockville, Md. 20852) under the ATCC designation number 69602.

EXAMPLE 13 Use of Recombinant Phage Anti-LDL ScFv Antibodies to DetectLDL

Intact phage displaying the recombinant anti-LDL ScFv antibody RcB₃M₁D₄were used in both ELISA and dipstick methods to detect LDL. ELISAmicrotiter plates were coated with a sheep antibody to M13 g8p coatprotein (Pharmacia Biotech Inc., Piscataway, N.J.) and blocked with a0.1% nonfat milk proteins as described above. After washing, therecombinant phage anti-LDL ScFv antibodies were added to the plate andallowed to bind to the anti-M13 g8p antibody overnight at 4° C. Becausethe anti-LDL ScFv antibody is expressed on the tip of the phage as aprotein fused to the minor coat g3p protein, the anti-LDL binding siteis free to bind LDL. Unbound recombinant phages were washed off and thewells incubated with dilutions of LDL (3 hours at room temperature). Theunbound LDL was then washed away, and the Pan B (D₆) MAbantibody-peroxidase complex was added and incubated as described above.Unbound Pan B (D₆) MAb was washed away and the chromogenic peroxidasesubstrate (ABTS) was added. The color intensity in each well was readusing an ELISA plate reader. The color intensity correlated with theconcentration of LDL used in each well of the plates. The negativecontrol wells coated with the native M13 phage showed no color at all.

The phage anti-LDL ScFv antibody RcB₃M₁D₄ was also used to explore itssuitability for the dipsticks. PVDF strips were sequentially coated withthe anti-g8p antibody and blocked with 2% nonfat milk proteins asdescribed above. This was followed by three sequential 24-hourincubations of the strips in a solution of RcB₃M₁D₄ phage antibody at 4°C. After washing in PBS, strips were incubated with LDL dilutionsfollowed by an incubation with the Pan B (D₆) MAb-peroxidase complex,washing, and incubation in TMB chromogenic substrate as described abovefor the HB₃cB₃ MAb dipsticks. The intensity of color on the phageanti-LDL ScFv antibody-coated dipsticks was proportional to theconcentration of LDL. The experiments with the phage anti-LDLdemonstrate that the recombinant anti-LDL ScFv antibodies are capable ofbinding to LDL under the conditions used in described ELISA as well asdipstick methods. Thus, anti-LDL ScFv antibodies made free of the phagecomponents (Hoogenboom, H. R. et al. Nucl. Acid. Res. 19:4133–4137(1991)) are likewise suitable, for use in methods and compositions suchas ELISA and dipstick methodologies described above.

The rest of the hybridomas described above, which which produce thecorresponding MAbs: AIbD₅, AIbE₂, CdB₅, XbA₃, EfB₁, EfD₃, are alsouseful to create a library of corresponding recombinant antibodies. Thisapproach offers several important advantages. ScFv-encoding DNArecombinant molecules have also been produced from cDNA of the AIbD₅ andD₆ hybridomas and can be inserted into pCANTAB5 for making recombinantantigen-specific antibodies such as the RcB₃M₁D₄ anti-LDL recombinantphage antibody described above.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

1. A method for determining the relative ratio of at least two differentlipoproteins or apolipoproteins in a biological sample comprising:immersing into the sample a solid phase material having separatelyimmobilized thereon at least first and second antibody molecules,wherein the antibody molecules are selected from the group consisting ofmonoclonal antibodies, recombinant antibodies and antigen-bindingantibody fragments thereof, wherein the antibody molecules areimmunoreactive with at least two different lipoproteins, wherein thefirst and second antibodies bind to different stable, conformationindependent epitopes that are uninfluenced by the lipid content of thelipoprotein, protein component of the lipoprotein or lipid associatedwith the specific lipoprotein, wherein the lipoproteins are selectedfrom the group consisting of LDL, HDL and VLDL; allowing the antibodymolecules time to bind to the LDL, HDL, VLDL or apolipoproteins in thesample; removing the solid phase material containing the immobilizedantibody molecules; determining the amount of lipoprotein orapolipoproteins bound by the immobilizedantibody molecules, andcomparing the amount bound which is specific for LDL, HDL, VLDL or eachapolipoprotein in order to calculate the relative amounts of LDL, HDL,VLDL or apolipoproteins.
 2. The method of claim 1 wherein the antibodymolecules immobilized on the solid phase material are immunoreactivewith lipoproteins selected from the group consisting of HDL and LDL. 3.The method of claim 2 wherein the antibodies to the HDL or LDL areselected from the group consisting of recombinant antibodies andantibody fragments.
 4. The method of claim 3, wherein the first orsecond monoclonal antibodies are the anti-LDL monoclonal antibodyproduced by the hybridoma cell line HB₃cB₃ ATCC designation number HB11612.
 5. The method of claim 3, wherein the first or second monoclonalantibodies are recombinant anti-LDL RcB₃M₁D₄ATCC designation number69602.
 6. The method of claim 1 further comprising determining theamount of lipoprotein lipid or lipid associating with apolipoprotein bystaining of the material bound to the immobilized antibody using a lipidstain.
 7. The method of claim 6 wherein the lipid stain is selected fromthe group consisting of Sudan Red 7B, Oil Red O, and Sudan Black B. 8.The method of claim 6 wherein the lipoprotein lipid is stained prior toimmersing the immobilized antibodies.
 9. The method of claim 6 furthercomprising measuring the amount of apolipoprotein or protein associatedwith the lipid in the sample, further comprising the step of providingantibodies immunoreactive with at least one apolipoprotein, wherein theantibodies are coupled to a protein stain, and staining theapolipoprotein or protein associated with the lipid in the sample byreacting the protein stain coupled antibodies with the apolipoprotein orprotein associated with the lipid in the sample.
 10. The method of claim1, wherein the apolipoprotein is selected from the group consisting ofApo A-I, Apo A-II, Apo B, Apo C-III, and Apo E.
 11. The method of claim1, wherein the biological sample is selected from the group consistingof blood, plasma, and serum.
 12. A method for determining the relativeratio of LDL to HDL in a biological sample comprising (a) determiningthe amount of LDL in the sample by adding to the sample monoclonalantibody molecules immunoreactive with low density lipoprotein and notcross-reactive with high density lipoprotein and determining the amountof low density lipoprotein; (b) determining the amount of HLD in thesample by adding to the sample monoclonal antibody moleculesimmunoreactive with high density lipoprotein and not cross-reactive withlow density lipoprotein and determining the amount of high densitylipoprotein; and (c) determining the ratio of the amount of low densitylipoprotein with the amount of high density lipoprotein, wherein atleast one of the monoclonal antibodies to LDL and HDL bind a stable,conformation independent epitope that is uninfluenced by the lipidcontent of the lipoprotein, the protein component of the lipoprotein orlipid associated with the specific lipoprotein.
 13. A method fordetermining the relative ratio of VLDL to HDL comprising (a) determiningthe amount of VLDL in the sample by determining the amount of Apo Epresent in the VLDL in the sample by providing Pan B antibody which ischaracterized by an equal binding and high affinity for all ApoB-containing lipoproteins in human plasma, providing monoclonal antibodywhich specifically binds to Apo E associated with VLDL, contacting theantibodies reactive with Apo E associated with VLDL with the biologicalsample to form complexes between the anti-ApoE antibodies and Apo Econtaining particles, contacting Pan B antibody with the biologicalsample containing the complexes between the anti-ApoE antibodies andApoE containing particles to form complexes of anti-ApoB-anti-ApoE-ApoEcontaining particles, and determining the amount of Apo E in thecomplexes of anti-ApoB-anti-ApoE-ApoE containing particles, which is theApo E present in VLDL in the sample; (b) removing the complexes ofanti-ApoB-anti-ApoE-ApoE containing particles, either by binding of theanti-Apo E antibodies to an immobilized surface or centrifugation ofsample to remove the complexes of anti-ApoB-anti-ApoE-ApoE containingparticles; and (c) determining the amount of HDL in the sample bydetermining the amount of Apo E present in the HDL in the sample byproviding Apo A-I monoclonal antibody immunoreactive specifically withApo A-I, contacting antibodies reactive with Apo E in HDL particles withthe biological sample to form complexes between the anti-ApoE antibodiesand Apo E containing particles, contacting the Apo A-I monoclonalantibody with the biological sample to form complexes of the anti-ApoEantibodies-ApoE containing particles-anti-ApoA-I, determining the amountof Apo E present in HDL in the complexes of the anti-ApoEantibodies-ApoE containing particles-anti-Apo A-I in the sample, anddetermining the ratio of Apo E present in VLDL in the sample and Apo Epresent in HDL in the sample which is the ratio of VLDL to HDL, whereinat least one of the monoclonal antibodies bind to a stable, conformationindependent epitope that is uninfluenced by the lipid content of thelipoprotein, protein component of the lipoprotein or lipid associatedwith a specific lipoprotein selected from the group consisting of Apo B,Apo AI, and Apo E.
 14. A kit for determining the relative ratio of VLDLto HDL comprising Pan B antibody which is characterized by an equalbinding and high affinity for all Apo B-containing lipoproteins in humanplasma, monoclonal or recombinant antibody specifically immunoreactivewith Apo C-III, and monoclonal or recombinant Apo A-I antibodyspecifically immunoreactive with Apo A-I, wherein at least one of themonoclonal or recombinant antibodies specifically bind to a stable,conformation independent epitope of a lipoprotein including Apo C-III orApo A-I that is uninfluenced by the lipid content of the lipoprotein,protein component thereof or lipid associated with a specificlipoprotein selected from the group consisting of Apo AI, and Apo CIII.15. The kit of claim 14 wherein the anti-Apo C-III or anti-A-Imonoclonal or recombinant antibody molecules are selected from the groupconsisting of monoclonal antibodies, recombinant antibodies, and antigenbinding antibody fragments thereof that specifically bind to a stable,conformation independent epitope which is uninfluenced by the lipidcontent of the lipoprotein, protein component thereof, or lipidassociated with a specific lipoprotein.
 16. A kit for determining therelative ratio of VLDL to HDL comprising Pan B antibody which ischaracterized by an equal binding and high affinity for all ApoB-containing lipoproteins in human plasma, monoclonal antibody whichbinds to Apo E associated with VLDL, monoclonal Apo A-I antibodyspecifically immunoreactive with Apo A-I, and monoclonal antibody whichbinds to Apo E in HDL, wherein at least one of the antibodies binds to astable, conformation independent epitope of a lipoprotein containing ApoE or Apo A-I that is uninfluenced by the lipid content of thelipoprotein, protein component of the lipoprotein or lipid associatedwith a specific lipoprotein.
 17. The kit of claim 16 wherein theanti-Apo E or anti-Apo A-I monoclonal antibody molecules are selectedfrom the group consisting of monoclonal antibodies, recombinantantibodies, and monoclonal antibody fragments.
 18. A kit for determiningthe relative ratio of LPA-I and LPA-II lipoprotein particles comprisingmonoclonal or recombinant Apo-A-I antibody specifically immunoreactivewith Apo A-I lipoproteins in human plasma; and monoclonal or recombinantApo A-II antibody specifically immunoreactive with Apo A-II, wherein theanti-Apo A-I or anti-Apo A-II monoclonal or recombinant antibodymolecules are selected from the group consisting of monoclonalantibodies, recombinant antibodies, and antigen-binding antibodyfragments thereof that specifically bind to a stable, conformationindependent epitope of a lipoprotein containing Apo A-I or Apo A-IIwhich is uninfluenced by the lipid content of the lipoprotein, proteincomponent of the lipoprotein, or lipid associated with a specificlipoprotein.
 19. The kit of claim 18 wherein the anti-Apo A-I andanti-Apo A-II monoclonal or recombinant antibody molecules are selectedfrom the group consisting of monoclonal antibodies, recombinantantibodies, and monoclonal antibody fragments that specifically bind toa stable, conformation independent epitope which is uninfluenced by thelipid content of the lipoprotein, protein component of the lipoprotein,or lipid associated with a specific lipoprotein.